Recent studies suggest that proinsulin connecting peptide (C-peptide) may show physiological functions in various tissues. This study was aimed to determine whether C-peptide could be involved in the regulation of lipolysis, adiponectin release, and function of mesenchymal stem cells (MSCs) in adipose tissue. Human subcutaneous adipose tissue was cultured in the presence of C-peptide and lipolysis was determined by glycerol measurement in the conditioned media. Effect of C-peptide on adiponectin secretion was evaluated in differentiated adipocytes. The adipogenic and osteogenic abilities of adipose MSCs were evaluated using Oil Red and Alizarin Red staining, respectively. C-peptide induced a significant decrease in basal lipolysis, a slight decrease (statistically insignificant) in isoproterenol-stimulated lipolysis, and a slight increase in adiponectin secretion at concentration of 8 nM, 16 nM and 4 nM, respectively. It had no effect on adipogenic and osteogenic differentiation of MSCs. However, at concentration of 4 nM, C-peptide significantly increased the proliferative capability of MSCs (p<0.05). These results suggest that C-peptide has physiological effects in human subcutaneous adipose tissue and contributes to the regulation of basal lipolysis and pool of MSCs.