Low-temperature storage has been widely used to extend the post-harvest life of many fruits. However, tolerance to cold is largely variable even among different fruit cultivars. In this regard, the banana cultivars Nanicão and Prata display different responses to low-temperature storage. Prata is less affected by cold storage, while the synthesis of volatiles is drastically reduced in Nanicão bananas. Therefore, this study aimed to investigate the effect of low temperature storage at the proteome level using a shotgun proteomics approach to identify a large number of proteins in these banana varieties. Seventy-four differentially expressed proteins were identified in cv. Prata and 43 in cv. Nanicão. Principal component analysis indicated that samples from control and cold storage groups could be differentiated based on their proteome. Overall, low temperatures decreased proteins related to carbohydrate metabolism and energy, defense, and transport in Nanicão bananas. Contrastingly, low temperatures induced the accumulation of proteins for carbohydrate metabolism and energy, transport, and defense in Prata bananas. Therefore, the observed changes in the proteome during cold acclimation and ripening in Nanicão and Prata bananas can explain, at least in part, the differences in low-temperature tolerance between these cultivars.