Human induced pluripotent stem cell (hiPSC)-derived endothelial cell (hiPSC-EC) transplantation is a promising therapy for treating peripheral artery disease (PAD). However, the poor differentiation of hiPSCs limits their clinical application. Therefore, finding key factors that regulate cellular differentiation is crucial for improving the therapeutic efficacy of hiPSC-EC transplantation. Sterol regulatory element binding protein 1 (SREBP1) is a key regulator of lipid metabolism and stem cell differentiation. However, it remains unknown whether SREPBP1 modulates hiPSC differentiation. In this study, we showed that SREBP1 expression was negatively associated with hiPSC differentiation and EC function. The results show that SREBP1 binds to the promoter region of miR199b-5p and suppresses its transcription, resulting in the activation of Notch1 signaling. Blocking SREBP1 increased both hiPSC differentiation and EC angiogenesis. These findings demonstrate a novel role for SREBP1 in hiPSC differentiation and EC angiogenesis.