BACKGROUND: Islet β-cell transplantation offers a promising treatment for repairing pancreatic damage in diabetes, with the transcription factor pancreatic duodenal homeobox-1 (PDX1) being crucial for β-cell function and insulin secretion. Mammalian threonine protein kinase (MST1) is recognized for its role in regulating PDX1 during cell apoptosis, yet its function in embryonic stem cell (ESC) differentiation into insulin-producing cells (IPCs) remain underexplored. This study investigated the effect of MST1-silencing on the differentiation of ESC into IPCs. METHODS: ESCs were transfected utilizing a recombinant MST1-silencing lentiviral vector (shMST1). qRT-PCR, immunofluorescence, flow cytometry, western blot and ELISA assays were performed to examine function of IPCs in vitro. Furthermore, these IPCs were transplanted into type 1 diabetic mellitus (T1DM) rats. Measuring the changes in blood glucose concentration of animals before and after IPCs transplantation. Intraperitoneal glucose tolerance test (IPGT) was used to determine the regulatory effect of IPCs transplantation on blood glucose stimulation and immunohistochemistry was used to detect the expression of pancreatic Insulin protein in T1DM rats. RESULTS: It was observed that IPCs from the shMST1 group exhibited notably improvement in insulin secretion and glucose responsiveness, suggesting MST1 suppression may enhance IPC maturity. The rats demonstrated significant normalization of blood sugar levels and increased insulin levels, akin to non-diabetic controls. This implies that MST1-silencing not only augments IPC function in vitro but also their therapeutic efficacy in vivo. CONCLUSIONS: The findings indicate that targeting MST1 offers a novel approach for deriving functionally mature IPCs from ESCs, potentially advancing cell replacement therapies for diabetes. This research underscores the importance of developing IPCs with competent insulin secretion for diabetes treatment in vitro.