Motilin is a circulating gastrointestinal peptide secreted primarily by duodenal mucosal M cells and recognized for its prokinetic effects on gastrointestinal tissues. Little information is available regarding effects on insulin/glucose homeostasis or adipocyte function. Our aim was to evaluate the effects of motilin on adipocyte proliferation, differentiation, lipolysis, and macronutrient uptake in adipocytes. 3T3-L1 cells and primary rat adipocytes were treated acutely and chronically with varying motilin concentrations, and effects were compared with vehicle alone (control), set as 100% for all assays. In preadipocytes, motilin stimulated proliferation ([(3)H]thymidine incorporation) and mitochondrial activity (141 ± 10%, P < 0.001 and 158 ± 10%, respectively, P < 0.001), in a concentration-dependent manner. Chronic supplementation with motilin during differentiation further increased lipogenesis (Oil red O staining 191 ± 27%, P < 0.05) and was associated with an upregulation of PPARγ (148 ± 8%, P < 0.01), C/EBPα (142 ± 17%, P < 0.05), and Cav3 (166 ± 20%, P < 0.05) expression. In mature 3T3-L1 adipocytes motilin increased fatty acid uptake/incorporation (≤ 202 ± 12%; P < 0.01) and glucose uptake (146 ± 9% P < 0.05) and decreased net fatty acid release (maximal -31%, P < 0.05) without influencing total lipolysis (glycerol release). Similar effects were obtained in primary rat adipocytes. Motilin acutely increased expression of PPARγ, CEBPβ, DGAT1, and CD36 while decreasing adiponectin mRNA and secretion. In human adipose tissue, motilin receptor GPR38 correlated with HOMA-IR and GHSR1 (r = 0.876, P < 0.0001). Motilin binding and fatty acid incorporation into adipocytes were inhibited by antagonists MB10 and [D-lys3]-GRP6 and PI 3-kinase inhibitor wortmannin. Taken together, these results suggest that motilin may directly influence adipocyte functions by stimulating energy storage.