Vaspin, a novel adipocyte factor secreted from visceral adipose tissues, is associated with obesity and insulin resistance and can regulate glucose and lipid metabolism, increase insulin sensitivity, and suppress inflammation; however, the underlying mechanisms remain unknown. Proliferation and maladaptive differentiation are important pathological mechanisms underlying obesity. This study aimed to evaluate the effects of vaspin on the proliferation and differentiation of preadipocyte 3T3-L1 cells and to explore the likely mechanisms responsible for 3T3-L1 differentiation. Vaspin was added to cultured 3T3-L1 cells, and the differentiation of adipocytes was evaluated using Oil Red O staining. The AKT signaling pathway and specific differentiation factors related to the differentiation of preadipocyte 3T3-L1 cells, peroxisome proliferator-activated γ and the CCAAT/enhancer-binding protein (C/EBP) family, were evaluated using reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses during the early phase of differentiation. Additionally, adiponectin mRNA, interleukin-6 mRNA (IL-6 mRNA), and glucose transporter-4 (GLUT4) protein levels were measured in the differentiated adipocytes. The results indicated that vaspin promotes the intracellular accumulation of lipids and increases differentiation-related factors, including peroxisome proliferator-activated receptor γ, C/EBPα, and free fatty acid-binding protein 4 (FABP4), in a dose-dependent manner. Additionally, vaspin (200 ng/mL) increased the mRNA and protein levels of C/EBPβ, peroxisome proliferator-activated γ, C/EBPα, and FABP4. Moreover, compared with the control, significantly smaller eight-day differentiated adipocytes were observed, and these cells exhibited decreased IL-6 mRNA and increased GLUT4 mRNA levels; these results also indicated the potential of vaspin to promote the insulin-mediated AKT signaling pathway during the early phase of differentiation. In conclusion, vaspin is able to promote the differentiation of 3T3-L1 preadipocytes and may increase their sensitivity to insulin and suppress obesity.