MicroRNAs are endogenous small non-coding RNAs that negatively regulate mRNAs, mainly at the post-transcriptional level, and play an important role in resistance response of plants. To date, there are few reports on resistance response of strawberry miRNAs to pathogens. In this study, using high-throughput sequencing, 134 conserved and 35 novel miRNAs were identified in six libraries within the treatment of Botrytis cinerea. A total 497 potential target genes were predicted using Fragaria vesca genome. Most of the differential expressed miRNAs in strawberry fruits were up-regulated in early libraries and down-regulated in late libraries. PIRL, the target gene of miR5290a, showed the opposite expressed trend compared with miR5290 from T1 to T3 libraries, and functional analysis of the PIRL gene shows that it has obvious resistance to B. cinerea in the strawberry fruits with overexpressed PIRL gene. We speculate that miR5290a negatively regulates its target gene PIRL to increase resistance to pathogen infection, and further analysis of PIRL function is meaningful for studying the plant-pathogen relationship and improving strawberry fruit quality and yield.