Adipocyte dysfunction is strongly associated with the development of insulin resistance and diabetes, and regulation of adipogenesis is important in prevention of diabetes. We prepared a 100% methanol fraction of methanolic extract from unripe kiwi fruit (Actinidia deliciosa), designated KMF (kiwi fruit methanol fraction). When applied to 3T3-L1 preadipocyte cells, KMF promoted adipocyte differentiation, increased glycerol-3-phosphate dehydrogenase (GPDH) activity, and increased triglyceride (TG) content. KMF markedly increased mRNA expression of peroxisome proliferator-activated receptor gamma (PPARgamma)-the master adipogenic transcription factor-and its target genes. Moreover, KMF increased mRNA expression and protein secretion of adiponectin, whereas mRNA expression and secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were decreased. Compared with troglitazone, KMF decreased the production of reactive oxygen species (ROS) and nuclear factor-kappaB (NFkappaB) activation. Glucose uptake was stimulated by KMF in differentiated 3T3-L1 adipocytes. Taken together, these results indicate that KMF may exert beneficial effects against diabetes via its ability to regulate adipocyte differentiation and function.