René Padilla was born in 1932 in Quito, Ecuador, the son of a tailor/evangelist and the sixth of eight children. The Padillas moved to Colombia in 1935, but they returned to Ecuador in 1944 due to the intense persecution of Protestants by the Roman Catholic majority in Colombia; they relocated in Quito, where René remained until 1953, when he came to the United States to study at Wheaton College. He had a conversion experience when he was 15, and subsequently became an active evangelist by participating in a jail ministry, radio preaching on radio station HCJB, and street preaching.

Padilla attended Wheaton College between 1954 and 1957, and continued in Wheaton's Graduate School, where he completed his masters degree in biblical studies in 1960. Although invited to the staff of International Fellowship of Evangelicals (IFES) in 1957, Padilla delayed his acceptance until he had completed his graduate work. Following his graduation in 1960, he was appointed to work as International Fellowship of Evangelical Student's (IFES) Latin American traveling secretary for Venezuela, Colombia, Ecuador and Peru. In 1961, he married Catherine Feser, and they settled in Bogota, Colombia. They eventually had four daughters and a son. In 1963, the Padillas moved to Manchester, England, where Padilla worked on his PhD in New Testament under F.F. Bruce.

He was appointed IFES's Associate General Secretary for Latin America in 1966 after completing his PhD, and the Padillas then returned to South America to Lima, Peru. Padilla worked among university students, overseeing the training of student leaders, and the development of campus and national movements throughout Latin America. In 1967, the Padillas moved to Buenos Aires, Argentina, to oversee IFES's literature work. In 1972, Padilla was appointed the director of IFES's publishing division, Ediciones Certeza. In 1973, Padilla became editor of IFES's magazine, Certeza.

Padilla worked with IFES until the end of 1981. In 1982 he joined the staff of Latin America Mission to become the associate editor of Latin America Mission Publications (Editorial Caribe) and develop Mission, a Spanish magazine. He was selected to be the General Secretary of the Latin American Theological Fraternity (an association of Latin American theologians somewhat like the Evangelical Theological Society) in 1984.

In addition to his work among students, Padilla authored books and articles, and participated in international and regional conferences, including the 1969 Latin American Congress on Evangelism, the 1974 Lausanne Congress, the 1980 International Simple Lifestyle Consultation, and the 1982 Consultation on the Relationship Between Evangelism and Social Responsibility. Padilla presented a plenary paper at the Lausanne Congress and was instrumental, along with other participants, in advocating a more active role among evangelicals in addressing social issues.

In 1973, Padilla became involved in a suburban church, the Evangelical Baptist Church of La Lucila; in 1976 he joined its pastoral team. The church developed ministry among slum dwellers and drug addicts, learning about and serving as a model for ministry to diverse human needs, fusing evangelism and social action.