Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.