Objective This study utilized an in vitro differentiation model of human embryonic stem cells （hESCs） into cardiomyocytes （CMs） to establish an early heart development model. It was intended to assess the embryotoxicity of perfluorooctanesulfonic acid （PFOS） and the potential protective effect of vitamin D （VitD）. Methods hESCs were induced to differentiate using differentiation media containing either PFOS alone or a combination of PFOS and VitD. At the terminal stage （day 8） of cardiomyocyte differentiation， the differentiation process was observed under an optical microscope. Quantitative real - time reverse transcription polymerase chain reaction （RT-qPCR） was employed to detect the expression of myocardial differentiation marker proteins ［cardiac troponin T（cTNT）， α-actinin， NK2 homeobox 5 （NKX2.5）］ and mitochondrial complexes ［ubiquinol-cytochrome c reductase core protein 1 （UQCRC1）， cytochrome c oxidase subunit 2 （MTCO2）， ATP synthase subunit alpha （ATP5A1）］ on the 8th day. Mitochondrial metabolism during differentiation was evaluated by measuring the ATP levels and reactive oxygen species produced by mitochondria on the 2nd day. Results The results clearly indicated that exposure to PFOS significantly inhibited the differentiation capacity of hESCs into CMs. This toxic effect was strongly associated with mitochondrial dysfunction， whereas VitD showed the potential to counteract this toxicity. In comparison with the control group， the density of mature cardiomyocyte colonies was notably reduced in the PFOS-exposed group. Nevertheless， in the group treated with both PFOS and VitD， the colony density was restored to a level approaching that of the control. Moreover， RT-qPCR results demonstrated that VitD effectively mitigated the PFOS-induced decrease in the expression of cardiomyocyte differentiation markers. Mitochondrial function tests further disclosed that PFOS exposure resulted in a substantial decline in mitochondrial energy metabolism efficiency in CMs derived from hESCs， triggered oxidative stress， and impaired normal mitochondrial function. These effects were reversed to normal levels after co-intervention with VitD. Conclusions The findings clearly demonstrated that PFOS inhibited the differentiation of hESCs into CMs. VitD could effectively counteract the toxic effects induced by PFOS and restore mitochondrial function via the regulation of mitochondrial-related signaling pathways.