Opus Dei began its apostolic activities in the United States in Chicago in 1949, when Sal Ferigle, a young physics graduate student, and Father Joseph Muzquiz, one of the first 3 priests to be ordained for Opus Dei, arrived in Chicago. The first center was established near the University of Chicago. (The cause of canonization of Father Muzquiz was opened in Boston in 2011 by Cardinal Sean O'Malley.)

Today there are more than 3,000 members in the United States, and a great many more people who participate in Opus Dei's activities of spiritual formation. These activities are organized from about 60 centers in a number of metropolitan areas, including: Boston; Chicago; Dallas-Fort Worth; Houston; Los Angeles; Miami; Milwaukee; New York City; Pittsburgh; Princeton, New Jersey; San Antonio; the San Francisco Bay area; South Bend, Indiana; St. Louis; Urbana-Champaign, Illinois; and Washington, D.C.

The Prelature of Opus Dei uses seven conference centers for retreats and seminars. They are located near Boston, Chicago, Houston, Miami, New York, San Francisco and Washington, D.C.

In addition to the personal apostolate that the Prelature fosters in its members and which occupies a pride of place, the Prelature as such also provides pastoral assistance to a number of educational and social initiatives. Supplementary educational programs at Midtown and Metro Centers, for example, have helped thousands of African-American and Hispanic youth from low-income families in Chicago.

Opus Dei members, in cooperation with others, operate eight secondary schools in the United States: The Heights and Oakcrest near Washington, D.C.; Northridge Preparatory School and Willows Academy near Chicago; the Montrose School and Sparhawk Academy near Boston; and Western Academy and Magnolia School near Houston. IESE Business School, the graduate business school of the University of Navarra, has a campus in Manhattan. Windmoor Study Center is residence for college men in South Bend, Indiana.

In 1991, Cardinal Joseph Bernardin, Archbishop of Chicago, entrusted the parish of St. Mary of the Angels to priests of Opus Dei. In 2016, Cardinal Timothy Dolan, Archbishop of New York, did likewise with the parish of St. Agnes, in Manhattan. The Catholic Information Center in Washington, D.C., and the Holy Cross Chapel in Houston, Texas, have also been entrusted to priests of Opus Dei.

Opus Dei's U.S. headquarters are located in New York City. Currently serving as Vicar for the United States is Father Javier del Castillo. Within the United States, there are two delegations: the delegation of Central United States (based in Chicago, with Father Peter Armenio as Vicar) and the delegation of California (based in Los Angeles, with Father Luke Mata as Vicar).