Background The physical features of substrate have attracted more and more attention in the in vitro culture of stem cells. Objective To explore the effect of concave and convex interfaceon in vitro culture of mouse embryonic stem cells. Methods Mouse embryonic stem cells were cultured on substrate with concave and convex interface.. The biological morphology of the cell colony was observed. The pluripotency of embryonic stem cells was detected by immunofluorescence and alkaline phosphatase staining. Results Embryonic stem cells on concave substrates and convex substrates have higher stereo and circularity than those on flat substrates, but it is more obvious on concave substrates.Besides , the expression level of Oct4-GFP and the staining intensity of alkaline phosphatase in embryonic stem cells which were cultured on concave substrates and convex substrates were significantly higher than those on flat basement, especially on concave substrates. Conclusion Compared with flat substrates, concave substrates and convex substrates have positive effects on the pluripotency maintenance of embryonic stem cells, which can help to maintain pluripotency, but concave substrates have better effects. The results showed that changing the curvature of the substrate can help maintain pluripotency of embryonic stem cells cultured in vitro. It is of great significance to the research and clinical application of embryonic stem cells.