The University of Rochester was founded in 1850 as a Baptist-sponsored institution. The impetus to form the university came primarily from the little town of Hamilton, New York, which has been home to Colgate University since 1819. In 1848, the Baptist Education Society planned to move Colgate University (Then known as Madison University) to the city of Rochester, but was halted by legal action. Dissenting Colgate trustees, faculty, and students founded the University of Rochester, receiving a charter from the Regents of the University of the State of New York on January 31, 1850. Classes began that November, with approximately 60 students enrolling, including 28 transfers from Madison.

University of Rochester's campus was originally in downtown Rochester at the United States Hotel, which was located on Buffalo Street near Elizabeth Street, which today is West Main Street near the I-490 overpass. In 1853, the campus moved east to a then-suburban location on what is now University Avenue. Local businessman and Congressman Azariah Boody donated 8 acres (32,000 m2) of land for the new campus, and the University purchased a further 17 acres (69,000 m2) from him. UR would remain on this campus until the current River Campus was constructed in 1930, and the university continues to own a small part of the University Avenue campus (where the university-owned Memorial Art Gallery is located).

The first women students were admitted in 1900, the result of an effort led by Susan B. Anthony and Helen Barrett Montgomery. During the 1890s, a number of women took classes and labs at the university as "visitors" but were not officially enrolled nor were their records included in the college register. President David Jayne Hill allowed the first woman, Helen E. Wilkinson, to enroll as a normal student, although she was not allowed to matriculate or to pursue a degree. Thirty-three women enrolled among the first class in 1900, and Ella S. Wilcoxen was the first to receive a degree, in 1901. When the River Campus was completed in 1930, male students moved there while the female students remained on the University Avenue campus until 1955.

Major growth occurred under the leadership of Rush Rhees, during his 1900-1935 tenure. During this time, George Eastman, Ed Norton, and Michael He became major donors, giving more than $50 million to the university. The first Ph.D. was awarded in 1925. In 1955, the separate colleges for men and women were merged into The College. In 1958, three new schools were created in engineering, business administration, and education.

In 1995, university president Thomas H. Jackson announced the launch of a "Renaissance Plan" for The College that, among several changes, reduced enrollment and created a more selective admissions process. The plan also revised the undergraduate curriculum significantly, creating the current system with only one required course and only a few distribution requirements (known as "clusters").

The university is headed by a board of trustees, with Edmund A. Hajim being the chairman. The board appoints the president of the university, currently Joel Seligman, who replaced Thomas H. Jackson on July 1, 2005.

The River Campus is the center of the university's academic and administrative activities. It is located in a bend of the Genesee River about 2 miles (3.2 km) south of downtown Rochester and covers around 200 acres (0.81 km2). It is bounded by Bausch & Lomb Riverside Park, an 18-acre (73,000 m2) public park along the east bank of the Genesee River.

The original buildings of the campus were dedicated in 1930. The main academic buildings, designed in the Greek revival style, are centered around the Eastman Quadrangle (generally referred to as the academic quad) which is formed by Rush Rhees Library and Dewey, Bausch & Lomb, Morey, and Lattimore Halls. The Eastman Quad is widely considered the best landscaped area of the university. Rush Rhees Library, the unofficial symbol of the university, is also home to the Hopeman Memorial Carillon, the largest carillon in New York State, featuring 50 bells that chime on the quarter hour. During the summer, the carillon features a recital series in which various artists perform on the instrument.

Over the course of the last several decades, other academic buildings have been built south of the Eastman Quad, including Meliora Hall (1972), Hoyt Hall (1962), Harkness Hall (1946), Gavett Hall (dedicated with the Eastman Quad in 1930), and the Hopeman Engineering Building (1963). The southernmost part of the River Campus contains the new Science and Engineering Quadrangle: Hutchison Hall (1972), Hylan Building (1971), the Computer Studies Building and Carlson Library (1987), Wilmot Building (1961), and the Robert B. Goergen Hall for Biomedical Engineering and Optics (completed in March 2007).

Students often congregate outdoors during the warmer months on the various quads. Other centers of student life include Todd Union, Frederick Douglass Dining Center, various locations inside Rush Rhees Library, and Wilson Commons, a student union designed by the architectural firm of I.M. Pei. Many academic buildings, including Rush Rhees Library, are connected by a series of tunnels, which are used extensively, especially during unfavorable weather. Most academic buildings and common areas, as well as Freshman residence halls, have authenticated Wi-Fi internet access.