Abstract: AIM:To investigate the roles of Wnt3a in differentiation of mouse embryonic stem cells (ESC) into cardiomyocytes. METHODS: Hanging drop culture was used to induce the differentiation of ESCs into cardiomyocytes through formation of embryoid bodies (EBs). Cardiac-specific protein TnT was detected by immunocytochemistry and Wnt3a and its inhibitor Dkk-1 were administered at different time points. The percentage of beating EBs was calculated at different time points. mRNA expression of Nkx2.5, GATA4 and β-MHC were detected by RT-PCR, and the protein expression of TnT was detected by Western blot. The untreated group, the Wnt3a-treated group during days 0 to 5 and the Wnt3a-treated group during days 5 to 10 were referred to as control group, D0-5 group and D5-10 group, respectively. The group treated with Dkk-1was referred to as Dkk-1 group. RESULTS: Spontaneously beating EBs were positively stained with TnT. Wnt3a treatment during EBs formation (days 0 to 5) produced higher percentage of beating EBs, higher gene expression of Nkx2.5, GATA4, β-MHC, ANP, and higher protein expression of TnT compared with those in control group, whereas Wnt3a treatment after EB formation (days 5 to 10) had the opposite results. Dkk-1 treatment during days 5 to 10 produced a higher percentage of beating EBs and higher protein expression of TnT compared with those in control group. Wnt3a treatment during days 0 to 5 plus Dkk-1 treatment during days 5 to 10 produced a higher percentage of beating EBs and higher protein expression of TnT compared with those treated with Wnt3a or Dkk-1 alone. CONCLUSION: Our results indicate that Wnt3a regulates the cardiac differentiation of ESCs in a time-dependent manner. The activation of Wnt3a during EB formation and the inhibition of Wnt3a after EB formation produce a higher degree of myocardial differentiation.