Based on the action of small molecule compounds, the efficiency of differentiation of mouse primary hepatocytes into insulin-producing cells (IPCs) was improved by changing the expression of miR-124 -2p. Hepatocytes were transfected with microRNA-124-3p (miR-124-3p) mimic or inhibitor, followed by a chemical-defined culture system for maturation of IPCs. Then, detect the expression of insulin-related genes and protein and insulin secretion of each stage during differentiation. The expression of Foxa2, PDX1, NeuroD, insulin1, and insulin2 in IPCs in the miR-124-3p inhibition expression group was significantly upregulated, while the results were opposite in the miR-124-3p overexpression group. The results of cell immunofluorescence and glucose stimulation in vitro of the miR-124-3p inhibition expression group showed that the expression of insulin, PDX1, and C-peptide was increased, and the differentiation efficiency was higher than those of the control group and overexpression group. The primary mouse hepatocytes were successfully reprogrammed into IPCs by small-molecule compounds. We found that miR-124-3p plays a negative regulatory role in the differentiation of hepatocytes into IPCs in vitro. Inhibition of miR-124-3p expression significantly increased the expression of FOXA2 and PDX1, promoted the differentiation of hepatocytes into IPCs, and increased the induction efficiency. (C) 2019 Elsevier Inc. All rights reserved.