Previously we have succeeded to isolate stem cells (HEAC) from human eyelid adipose tissue, and functionally differentiate them into insulin-secreting cells. In the present study, we examined whether insulin family members might affect the insulinogenic differentiation of HEAC. Insulin treatment during culture affected little on the insulin and c-peptide secretions from HEAC after culture. However, insulin-like growth factor (IGF) 1 treatment decreased both secretions, whereas IGF2 greatly increased the secretions in a glucose-dependent manner. HEAC treated with IGF2 showed stronger expression of Pdx1, Isl1, Pax6 and PC1/3 genes compared to the control. They also showed distinct staining with insulin and c-peptide antibodies, and dithizone. While insulin or IGF2 treatment increased total cell number by 1.3- or 1.5-fold, respectively, each treatment increased the amount of insulin secretion by 27.1- or 78.1-fold, respectively. IGF2-enhanced insulinogenic differentiation was completely blocked by an antibody against insulin receptor (IR), but not by an antibody against IGF1 receptor (IGF1R). Differentiated HEAC showed expression of both IR and IGF1R genes while they expressed neither IGF2 nor IGF2R genes. Based upon these results, it is suggested that whereas IGF1 might inhibit the insulinogenic differentiation of HEAC, insulin and IGF2 could enhance the differentiation, and that the enhancing effect could be mediated via IR.