1) To investigate the effect of FOXC2 on the differentiation of adipose-derived mesenchymal stem cells. 2) To analyze the mechanism between FOXC2 expression regulation in adipose differentiation and insulin resistance (IR). We first amplified the FOXC2 promoter region-512 and cloned it into the luciferase expression vector. The reporter gene system was transfected into the adipose tissue-derived mesenchymal stem cell to study insulin-mediated FOXC2 expression. We also manipulated FOXC2 protein expression by either siRNA or overexpression and studied the differentiation capability of adipose tissue-derived mesenchymal stem cell into adipocytes, as well as the influence on several IR-related genes: GLUT4, PPARγ, UCP1 and PAI-1. 1) Insulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation (P<0.01). Insulin also induced FOXC2-pro-512T promoter activity significantly (P<0.01). 2) The stem cell adipose differentiation decreased in the FOXC2 overexpression group. 3) When FOXC2 was overexpressed, the expression of GLUT4, PAI-1 and UCP1 was higher than control groups (p<0.001). When FOXC2 was down-regulated by siRNA, both GLUT4 and PAI-1's protein expression were decreased (p<0.001), and the protein expression of PPARγ was increased (p<0.001). In the presence of insulin induction, overexpression of FOXC2 led to significantly higher UCP-1 expression (p<0.001) and lower PAI-1 expression (p<0.001). The protein expression of GLUT4, PAI-1 (p<0.001) and UCP-1 (p<0.05) was decreased in cells transfected with FOXC2 siRNA. Insulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation, possibly through the regulation of the FOXC2-pro-512T promoter activity. The different protein expression of FOXC2 has regulatory effects on several genes related to insulin resistance. FOXC2 is an important regulatory factor in adipocyte differentiation and insulin resistance. © 2020 Zhang et al.