Objective To investigate the role of different culture densities of embryoid bodies (EBs) in cardiac dif-ferentiation of mouse embryonic stem cells (ESCs). Methods The mouse ESCs were cultured in hanging drops for 3 days, followed by another 2 days for suspension culture to form EBs. Suspended EBs of different densities (60 or 120 EBs/60 mm tissue culture dish) were transferred onto tissue culture plates. The cardiac specific troponin T (TnT) was detected by immu-nocytochemistry. The percentage of beating EBs was calculated at different time points. The mRNA expression of cardiac spe-cific transcriptional factors Nkx2.5, GATA4 and cardiac specific proteinsβ-MHC and ANF were detected by RT-PCR. The protein expressions of TnT and p38 were detected by Western-blot assay. Results Spontaneously beating EBs were posi-tively stained with TnT. There were significantly higher percentage of beating EBs, higher gene expression levels of Nkx 2.5, GATA4,β-MHC and ANF and higher protein expression of TnT in high density culture group than those of low density cul-ture group (P＜0.05). Furthermore, there was significantly higher activity of p38 pathway in high density culture group than that of low density culture group. And the percentages of beating EBs and TnT protein expression were decreased by p 38 pathway inhibitor SB203580. Conclusion The culture density of EBs is important in regulating the cardiac differentiation of ESCs. The high cell-density density culture of EBs enhances the cardiac differentiation of ESCs, which might be mediated by p38 signaling pathway.