Abstract: Strawberry is an important and highly economic crop worldwide, with the genomes of both its wild-type and cultivated varieties having been fully sequenced. Vacuolar processing enzyme (VPE), a class of cysteine proteases, play a critical role in the processing and activation of vacuolar proteins in plants. They are also implicated in inducing vacuole rupture and initiating proteolytic cascades that mediate programmed cell death. Despite their importance, research on the VPE gene family in strawberry remains limited. In this study, strawberry VPE genes were identified on the level of whole genome and analyzed on its molecular characterization. A total of 54 strawberry VPE genes were identified through phylogenetic analysis, all of which were distributed in plant vesicles, and exhibited diverse structures but shared a conserved Peptidase_C13 structural domain. The results showed that cis-acting regulatory elements were identified in cultivated strawberries, which were absent in wild types, suggesting that some VPE genes in cultivated strawberries may have undergone independent evolution. Molecular characterization of the strawberry VPE gene family revealed its functional role in vacuolar-mediated programmed cell death. These findings lay the foundation for exploring the regulation techniques of strawberry VPE proteins in strawberry growth and fruit quality, as well as the molecular mechanisms of stress response in the future.