Previous studies have shown that miR-322 regulates the functions of various stem cells. However, the role and mechanism of embryonic stem cell (ESCs) differentiation into cardiomyocytes remains unknown. Celf1 plays a vital role in stem cell differentiation and may be a potential target of miR-322 in ESCs' differentiation. We studied the function of miR-322An using mESCs transfected with lentivirus-mediated miR-322. RT-PCR results indicated that miR-322 increased NKX-2.5, MLC2V, and α-MHC mRNA expression, signifying that miR-322 might promote the differentiation of ESCs toward cardiomyocytes in vitro. The western blotting and immunofluorescence results confirmed this conclusion. In addition, the knockdown of miR-322 expression inhibited ESCs' differentiation toward cardiomyocytes in cultured ESCs in vitro. Western blotting results showed that miR-322 suppressed celf1 protein expression. Furthermore, Western blotting, RT-PCR, and immunofluorescence results showed that celf1 may inhibit ESCs' differentiation toward cardiomyocytes in vitro. Overall, the results indicate that miR-322 might promote ESCs' differentiation toward cardiomyocytes by regulating celf1 expression.