In his second version of the Conversion of St Paul for the Cerasi Chapel (1601), Caravaggio emphasizes darkness – not light – as an essential condition of mystical union. Caravaggio’s darkness, typically interpreted as a signifier of ‘realism’, possesses a metaphorical quality that goes well beyond mere tenebrism. Through an analysis of the interrelated themes of darkness and blindness in Caravaggio’s Conversion of Saint Paul, this paper suggests that the artist drew inspiration from Negative Theology, a strand of Neoplatonism that describes the path to the divine as one of increasing darkness. In looking at this painting within the context of seventeenth-century mysticism and its texts (Pseudo-Dionysius and St John of the Cross especially), it is possible to interpret Caravaggio’s use of darkness as an experimental dialectic on Negative Theology. A comparative analysis of Caravaggio’s two versions of Paul’s conversion (the earlier one was rejected) illustrates his radical reinterpretation of Pauline iconography and an increased emphasis on themes of darkness and negation. Given the profound stylistic change represented by Caravaggio’s revised composition – a picture completed only months after the first one was rejected – the Neoplatonic overtones in the second version very likely stem at least partially from suggestions from Caravaggio’s patrons. The Augustinian Fathers of the Madonna della Consolazione, responsible both for the rejection and re-commissioning of Caravaggio’s Cerasi laterals, were well versed in the Neoplatonic teachings of St Augustine and his insistence on the ineffability of the divine. Caravaggio’s dramatic stylistic shift towards darkness represents a sophisticated response to the changing ways in which spiritual conversion was conceived in the seventeenth-century Catholic Church.