Castration of male pigs is a common practice used to reduce boar taint in commercial pork production, but it also significantly results in fat accumulation in carcass. Our previous study revealed a miRNA gene, ssc-miR-7134-3p that was implicated in adipogenesis. However, the relationship between ssc-miR-7134-3p and fat deposition due to castration is unknown. In the present study, we observed that ssc-miR-7134-3p targets the coding sequence (CDS) region of MARK4 based on bioinformatics analysis and dual-luciferase assays. Experiments using silent mutations and sub-cloning showed that ssc-miR-7134-3p binds independently to two adjacent sites in the MARK4 CDS. Subsequently, ssc-miR-7134-3p inhibits MARK4 protein expression in pig fibroblast cells, being consistent with the targeting demonstrated in vitro. We found higher MARK4 protein levels in the back fat of castrated pigs than in intact pigs, providing further evidence that MARK4 is involved in regulation of fat deposition. In addition, one SNP (g. 2581A>G) in MARK4 was significantly associated with the back fat trait in Chinese and European pig populations. Taken together, we would conclude that ssc-miR-7134-3p targets the MARK4 gene for fat accumulation in the castrated male pigs.