Objective To explore the effect of concave and convex interface on 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 cell colony was observed. The pluripotency of embryonic stem cells was detected by immunofluorescence and alkaline phosphatase (ALP) staining. Results Embryonic stem cells on concave substrates and convex substrates had higher stereo degree and circularity than those on flat substrates, but it was more obvious on concave substrates. Besides, the expression level of Oct4-GFP and the staining intensity of ALP 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 sCompared with flat substrates, concave substrates and convex substrates had positive effects on the pluripotency maintenance of embryonic stem cells, which could help to maintain pluripotency, but concave substrates had better effects. Changing the substrate curvature could help to maintain pluripotency of embryonic stem cells cultured in vitro. The research findings are of great significance to the study and clinical application of embryonic stem cells.