Second in seniority of the orders of regular clerics (the Theatines being first),[2] the Barnabites were founded in Milan, by Anthony Mary Zaccaria, Barthélemy Ferrari, and Jacopo Antonio Morigia.

[2] The region was then suffering severely from the wars between Charles V and Francis I, and Zaccaria saw the need for radical reform of the Church in Lombardy, afflicted by problems typical for that era: dioceses without a bishop, clergy with inadequate theological training, a decrease in religious practice, and monasteries and convents in decline.

[2] In 1538 the grand old monastery of Saint Barnabas by the city wall of Milan was given to the congregation as their main seat, and thenceforth they were known by the popular name of Barnabites.

[4] After the death of Zaccaria in 1539, the congregation was favoured and protected by Archbishop Charles Borromeo of Milan and later by Francis de Sales because of their successful missionary work in Upper Italy.

In an address in 2000, to the institute's General Chapter, Pope John Paul II noted, "[I]n pointing out the ideal of religious and apostolic life to his spiritual sons, St Anthony Mary Zaccaria emphasized charity.

[2] The focus of the goals of the Barnabite Order, besides preaching in general, catechizing, hearing confessions, giving missions, ministrations in hospitals and prisons, and the education of youth, includes also a particular devotion to the thorough study and exposition of St. Paul's Epistles.

Many Barnabites became great scholars and scientists, including the astronomers Redento Baranzano and Paolo Frisi, the naturalist Ermenegildo Pini and the meteorologist Francesco Denza.

John Bellarini (1552–1630), who was the Visitor of the Order, and twice held the office of Assistant Superior General, was also a theologian who wrote a number of works including an influential commentary[24] on the Council of Trent.