The first service in the church building, designed by Scottish architect John Notman, was held on March 27, 1859.

[2] The church's rector from 1862 to 1869 was Phillips Brooks, who was also the author of the lyrics to the familiar Christmas carol "O Little Town of Bethlehem".

After the end of the American Civil War, Brooks took a sabbatical from the church to travel to Europe, Israel and Palestine.

His visit to Bethlehem inspired him to write a poem for his Sunday School students, and for the church's 1868 Christmas season he had church organist Louis Redner set the poem to music.

Philadelphia's Church of the Holy Trinity is also known for its numerous stained-glass windows, including five by Louis Comfort Tiffany and one by Luc-Olivier Merson.