Prothioconazole is a fungicide that has been widely used in general agriculture and livestock husbandry. This study evaluated the acute toxicity of prothioconazole to zebrafish embryos by assessing their hatching rate and malformation when exposed to different concentrations of prothioconazole. The 96 h-LC value of zebrafish embryos was 1.70 mg/L. Upon exposure to 0.85 mg/L, the mortality rate of the embryos significantly increased while their hatching rate decreased significantly. At prothioconazole concentrations higher than 0.43 mg/L, developmental morphologic abnormalities such as heart and yolk-sac edema, spine curvature, tail deformity, shortened body length and decreased eye area were observed. The heart rate of embryos decreased in a dose-dependent fashion during the exposure time. Prothioconazole exposure also resulted in increased rates of cardiac malformation detected by significant increase in the distance between the sinus venosus and bulbus arteriosus and the pericardium area. Moreover, the expression levels of genes related to cardiac development (amhc, vmhc, fli1, hand2, gata4, nkx2.5, tbx5 and atp2a2a) were significantly altered after exposure to prothioconazole. Indeed, this study revealed the adverse effects on the developmental and cardiovascular system of zebrafish embryo caused by prothioconazole. It further elucidated the risk of prothioconazole exposure to vertebrate cardiovascular toxicity. As such, it provides a theoretical foundation for pesticide risk management measures.