MicroRNAs are endogenous ~22nt RNAs that negatively regulate gene expression at the posttranscriptional level via binding to the 3′-untranslated region (3′UTR) of target mRNAs. The microRNA miR-27a was reported to depress the expression of myostatin, a critical inhibitor of skeletal myogenesis, by binding to its 3′UTR in mouse. In this study, we cloned the full-length 3′UTR of porcine myostatin by rapid amplification of 3′-cDNA ends (3′-RACE) and demonstrated that the 3′UTR of porcine myostatin is targeted by miR-27a. The phenomenon that the level of myostatin inversely correlated with miR-27a was observed in fat and heart of pigs and also in proliferating porcine myoblasts. Besides, overexpression of miR-27a in porcine myoblasts promoted cell proliferation by reducing the expression of myostatin. Our data suggest that miR-27a positive regulates porcine myoblast proliferation via targeting myostatin.