Intramuscular fat (IMF) content affects the tenderness, juiciness, and flavor of pork. An increasing number of studies are focusing on the functions of microRNAs (miRs) during porcine intramuscular preadipocyte development. Previous studies have proved that miR-425-5p was enriched in porcine skeletal muscles and played important roles in multiple physiological processes; however, its functions during intramuscular adipogenesis remain unclear. To explore the role of miR-425-5p in porcine intramuscular adipogenesis, miR-425-5p agomir and inhibitor were used to perform miR-425-5p overexpression and knockdown in intramuscular preadipocytes, respectively. Our results showed that the agomir of miR-425-5p dramatically inhibited intramuscular adipogenic differentiation and downregulated the expression levels of adipogenic marker genes PPARγ, FABP4, and FASN, whereas its inhibitor promoted adipogenesis. Interestingly, the agomir repressed proliferation of porcine intramuscular preadipocytes by downregulation of cyclin B and cyclin E. Furthermore, we demonstrated that miR-425-5p inhibited adipogenesis via targeting and repressing the translation of KLF13. Taken together, our findings identified that miR-425-5p is a novel inhibitor of porcine intramuscular adipogenesis possibly through targeting KLF13 and subsequently downregulating PPARγ.