The Jesuits first arrived in the Paraná area during the late sixteenth century. Along with the official Church and other regular orders, they enjoyed favourable tax concessions and initially prospered under the protection of the Crown. The first missions to the Guaraní were established in the upper Paraná from 1609. Though the Jesuits were to try to evangelize other parts of the country, such as the Chaco and northern Patagonia, over the next 150 years, it was in the subtropical Upper Paraná where they had their greatest success. After raids in the region by roaming Portuguese slavers in the 1630s, the Jesuits established their own indigenous militias for protection. Thereafter, Jesuit activity thrived: there were as many as thirty missions here by the beginning of the eighteenth century, with a total indigenous population exceeding 50,000. The Guaraní who lived in the missions had the benefit of Jesuit education and skills, and were exempt from forced labour in silver mines, but this was no earthly paradise: coercion and violence were not unknown, and epidemics ravaged these densely populated communities on a periodic basis.

In the seventeenth century, the Crown revoked its tax concessions to the Jesuits and their communities were forced to enter the colonial economy. They did so with characteristic vigour, becoming exporters of yerba mate , sugar and tobacco. However, their success aroused jealousy. Some missions housed more than 4000 indigenous people and this monopolization of labour , together with the economic and political influence of the Jesuits in Córdoba, stirred the resentment of nearby settlers. In the 1720s, secular settlers rebelled, urging the Crown to curb the domination of the Jesuits. In and around Asunción and Corrientes, they subjected Jesuit communities to raids , kidnappings, massacres and starvation. Though it survived that threat, the mission experiment was to succumb soon afterwards, a victim of power politics in Europe, lobbying from landed interests bent on exploiting the native labour, and the Bourbon King, Charles III's perception that the existence of a powerful Jesuit community answerable to the pope was a threat to secular royal authority. In 1767, he ordered their expulsion from all Spanish territories - an order carried out the following year, as captured dramatically in the film, The Mission , starring Robert de Niro. The remaining communities were subsequently entrusted to the Franciscans, whose mismanagement and neglect led to them being plundered and to many Guaraní being led away to slavery.