He attended the minor and major seminary of the archdiocese of Florence and was ordained a priest on 29 June 1966 in Florence. He served as vicar in the parish of St. Michael in San Salvi. In 1968 he became assistant at the minor seminary of Florence and then rector in 1972. Seven years later, he was subsequently appointed rector of the major seminary of Florence. He served there for 11 years and was then appointed pro-vicar and vicar general of the archdiocese.
On 22 February 2014, Bassetti was raised to the rank of cardinal by Pope Francis. Bassetti commented in response: "My aims have not changed. I want to visit factories and hospitals, because as the Pope says, we must 'be pastors with the odor of sheep.' This is the time to roll our sleeves back." He also praised the Pope's nomination of Archbishop Loris Capovilla as "a direct reference the Second Vatican Council". La Stampa called it a "surprise nomination". It was the first time a bishop of Perugia had been named a cardinal since Gioacchino Pecci, later Pope Leo XIII, in 1853. His selection was seen, according to John L. Allen, as a demonstration of Francis' preference for "traditionally neglected locales" and for "political moderates". Another observer described him as "instantly papabile, a man cut from the same cloth as Francis". Giovanni Paciullo, Rector of the University for Foreign Students of Perugia, called the nomination a tribute to Bassetti's "constant concern for the least, the excluded, the foreigner... which brought him into the paths of marginalization and suffering..." Bassetti has been a strong proponent of the traditional Tridentine Latin mass. Pope Francis named him a member of Congregation for Bishops, Congregation for the Clergy, and Pontifical Council for Promoting Christian Unity. In 2016 at the request of Pope Francis he authored the meditations for the Good FridayWay of the Cross service at the Colosseum. On 23 May 2017, Pope Francis named Bassetti to a five-year term as head of the Italian Episcopal Conference. Bassetti's name was one of three presented to the Pope by the Conference membership and the one who had received the most votes from them. At the same time he extended Bassetti's appointment as Archbishop of Perugia for five years despite the fact that he had submitted his resignation as required on his 75th birthday a few weeks earlier. Opening the CEI's annual meeting on 25 September, he called for collegiality and dialogue between bishops and with the entire church. He said he planned to end the practice of beginning the annual meeting with the president's address and would instead conclude the meeting by summarizing their debate and "seeking a point of equilibrium". As a starting point, he said Catholics could not be divided into those who prioritize personal morality and those who advocate for social justice. He spoke of the need to integrate migrants into Italian society, implying support for citizenship for those born in Italy. A reform of the tax system, he said, could support families, especially large ones, promote childbirth and provide employment. Pope Francis named him a member of the Congregation for the Oriental Churches on 6 August 2019.
