St. Ann & the Holy Trinity was built as The Church of the Holy Trinity by Brooklyn paper manufacturer Edgar Bartow, who wanted a magnificent edifice for the City of Brooklyn with pews that were rent-free. Bartow chose architect Minard Lafever to design the church and adjoining chapel and rectory, which are considered to be the finest achievement of Lafever's career. An important example of Gothic Revival architecture in America, the richly ornamented church is notable for its elaborately vaulted roof and extensive suite of stained glass windows by William Jay Bolton. St. Ann's pipe organ was built in 1925 by Ernest M. Skinner (Opus #524), and is the third instrument to be installed since the church opened in 1847.

During the Cold War in the 1950s, there was dissension within the congregation which eventually led to the dissolution of the church in 1957. The building was closed and stood mostly vacant for more than a decade. In 1969, nearby St. Ann's Church, the oldest Episcopal congregation in Brooklyn moved four blocks into the Holy Trinity building. St. Ann's took the new name of St. Ann & the Holy Trinity in honor of the building's heritage.

Today's parishioners continue the life of a body of faithful Episcopalians who have worshiped in Brooklyn for more than 240 years, first gathering during the American Revolution. Bishop Lawrence Provenzano designated the church as pro-cathedral during a festive Evensong service at St. Ann & the Holy Trinity on September 16, 2018.