Son to a family of musicians, he became an apprentice to Denis Calvaert's Bolognese studio at the age of nine, soon after having Albani and Domenichino as his colleagues. At the age of twenty, the three alumni migrated to the Accademia degli Incamminati (Academy of the newly embarked), a rising rival studio, under the teachings of Ludovico Carracci. They formed a school of prolific and successful Bolognese painters.

Reni left the academy after an argument over unpaid work, in 1598, through this time, he completed commissions for his altarpieces and his first series of prints, commemorating Pope Clement's visit to Bologna.

In 1601, Reni moved to Rome along with Albani, led by Ludovico's Cousin, Annibale Carracci, to work in fresco decorations of the Farnese Palace, till 1604, under the patronage of Cardinal Paolo Emilio Sfondrati, mainly. The next year he received an independent commission to make an altarpiece of the Crucifixion of St. Peter. He was one of the painters most sponsored by the Borghese family, from 1607 to 1614, becoming one of the premier painters during Paul V's papacy.

While in Rome, he accomplished his painting of the Archangel Michael, in 1630, for the church of Santa Maria Della Concezione dei Cappuccini, commissioned by Pope's Urban VIII's family, the Barberini. He also frescoed the Aldobrandini wings of the Vatican as well the Paoline Chapel of Santa Maria Maggiore, in Rome.

Often considered his fresco masterpiece, Reni's painted the ceiling of the large central hall of the Casino dell aurora, located on the Palazzo Pallavicini-Rospigliosi. This masterpiece, named Aurora (Dawn), depicts Apollo in his Chariot, preceded by Aurora bringing light to the world. That represents a characteristic of Reni's works, exploring myth-historic themes.

The artist had many pupils. Probably the most distinguished of them was Simone Cantarini. Another noteworthy pupil of his was Antonio Randa, a promising alumn until he tried to kill his very teacher.

Some of the artist's most noteworthy artworks are Ecce Homo, St. Joseph with the Infant Jesus, Drinking Bacchus, and Archangel Michael, to name only a few since his oeuvre his quite vast.

Guido Reni died in 1642, in Bologna, buried at the Basilica of San Domenico.