Recent studies suggest that proinsulin-connecting peptide (C-peptide) may exhibit characteristics of a hormone and 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. The level of 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. The tetrazolium bromide test was conducted for evaluating the effect of C-peptide on MSCs proliferation. C-peptide induced a significant decrease in basal lipolysis at concentrations of 8 and 16 nM (p < 0.05). It had no significant effects on isoproterenol-stimulated lipolysis, adiponectin secretion, and adipogenic or osteogenic differentiation of MSCs. At a concentration of 4 nM, this peptide significantly increased the proliferative capability of MSCs (p < 0.05). These results suggest that C-peptide has some physiological effects in human subcutaneous adipose tissue and contributes to the regulation of basal lipolysis and pool of MSCs.