Papaya (Carica papaya) is a trioecious species, with female, male, and hermaphrodite plants. Because of sex segregation, selecting hermaphroditic plants is vital for orchard establishment due to their higher commercial value. In addition to the costly sexing step, environmental stresses can result in abnormal flower development. However, molecular mechanisms that regulate sex differentiation in papaya are still poorly known. Thus, this study aimed to identify proteins associated with sex development in female and hermaphrodite flowers of papaya through comparative proteomic analysis. Proteins from flower buds at the early and late developmental stages of three papaya genotypes (UENF-CALIMAN 01, JS12, and Sunrise Solo 72/12) were studied via proteomic analysis via the combination of the shotgun method and nanoESI-HDMSE technology. In buds at an early stage of development, 496 proteins exhibited significantly different abundances between sexes for the SS72/12 genotype, 139 for the JS12 genotype, and 165 for the UC-01 genotype. At the final stage of development, there were 181 for SS72/12, 113 for JS12, and 125 for UC-01. The large group of differentially accumulated proteins (DAPs) between the sexes was related to metabolism, as shown by the observation of only the proteins that exhibited the same pattern of accumulation in the three genotypes. Specifically, carbohydrate metabolism proteins were up-regulated in hermaphrodite flower buds early in development, while those linked to monosaccharide and amino acid metabolism increased during late development. Enrichment of sporopollenin and phenylpropanoid biosynthesis pathways characterizes hermaphrodite samples across developmental stages, with predicted protein interactions highlighting the crucial role of phenylpropanoids in sporopollenin biosynthesis for pollen wall formation. Most of the DAPs played key roles in pectin, cellulose, and lignin synthesis and were essential for cell wall formation and male flower structure development, notably in the pollen coat. These findings suggest that hermaphrodite flowers require more energy for development, likely due to complex pollen wall formation. Overall, these insights illuminate the molecular mechanisms of papaya floral development, revealing complex regulatory networks and energetic demands in the formation of male reproductive structures.