Apart from the bandeirantes, the most important agents of the colonization of the interior were the Jesuits . The first Jesuit missionaries arrived in Brazil in 1549 and, thanks to the influence they held over successive Portuguese kings, they acquired power in Brazil second only to that of the Crown itself. In Salvador they built the largest Jesuit college outside Rome, and set in motion a crusade to convert the Indian population. The usual method was to congregate the Indians in missions , where they worked under the supervision of Jesuit fathers. From 1600 onwards, dozens of missions were founded in the interior, especially in the Amazon and in the grasslands of the Southeast.

The role the Jesuits played in the conversion of the Indians was ambiguous. Mission Indians were often released by Jesuits to work for settlers, where they died like flies; and the missionaries' intrepid penetration of remote areas resulted in the spread of diseases that wiped out entire tribes. On the other hand, many Jesuits distinguished themselves in protecting Indians against the settlers, a theological as well as a secular struggle, for many Portuguese argued that the native population had no souls and could therefore be treated like animals.

The most remarkable defender of the Indians was Antônio Vieira , who abandoned his position as chief adviser to the king in Lisbon to become a missionary in Brazil in 1653. Basing himself in São Luís, he struggled to implement the more enlightened Indian laws that his influence over King João IV had secured, to the disgust of settlers clamouring for slaves. Vieira denied them for years, preaching a series of sermons along the way that became famous throughout Europe, as well as Brazil: "An Indian will be your slave for the few days he lives, but your soul will be enslaved for as long as God is God. All of you are in mortal sin, all of you live in a state of condemnation, and all of you are going directly to Hell!" he thundered from the pulpit in 1654, to the fury of settlers in the congregation. So high did feelings run that, in 1661, settlers forced Vieira onto a ship bound for Portugal, standing in the surf and shouting "Out! Out!"

But Vieira returned, with renewed support from the Crown, and Jesuit power in Brazil grew. It reached a peak in the remarkable theocracy of the Guaraní missions , where Spanish and Portuguese Jesuits founded over a dozen missions on the pampas along the Uruguayan border. Left alone for the first fifty years, they effectively became a Jesuit state, until the Treaty of Madrid in 1752 divided up the land between Spain and Portugal; the treaty ordered the missions abandoned, so that settlers could move in. The Guaraní revolted immediately and, while the Jesuit hierarchy made half-hearted efforts to get them to move, most of the priests stayed with their Guaraní flocks. Resistance was heroic but hopeless: the superior fire power of a joint Spanish-Portuguese military expedition decimated both Guaraní and Jesuits in 1756.

Jesuit involvement in the Guaraní war lent added force to the long-standing settler demands to expel them from the colony. This time, they were helped by the rise to power of the Marquis de Pombal , who became the power behind the Portuguese throne for much of the eighteenth century. Seeing the Jesuits as a threat to Crown control, he seized upon the Guaraní wars as an excuse to expel the Order from Brazil in 1760. The Jesuits may have been imperfect protectors, but from this time on the Indians were denied even that.