The mechanism regulating procyanidin (PA) accumulation in banana (Musa acuminata) fruit is not understood. During this study, the effects of PA treatment on the activities of banana PA biosynthetic enzymes and transcriptomic profiles were investigated. The results showed that PA treatment delayed the decreases in leucoanthocyanidin reductase and anthocyanidin reductase activities, which affected the accumulation of PA. Furthermore, the peel samples of the control fruit and the PA-treated fruit on day 1 were selected for transcriptomic analysis. The results revealed that PA treatment induced 1086 differentially expressed genes. Twenty-one key genes, including those encoding biosynthetic enzymes and regulatory factors involved in PA biosynthesis, were validated using a quantitative real-time polymerase chain reaction. The results showed that these genes were upregulated by PA treatment during banana storage. Taken together, our study improves current understanding of the mechanism underlying PA-regulated banana senescence and provide new clues for investigating specific gene functions.