The Forkhead transcription factor O 1 (FoxO1) gene plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes clonal cell line differentiation. However, tissue expression of porcine FoxO1 and its function during porcine preadipocyte differentiation remained poorly understood. In the present study, we investigated tissue expressions of FoxO1 in pig by real time quantitative RT-PCR (qRT-PCR) and western blotting, and explored its role in porcine preadipocytes differentiation by RNA interference technique and qRT-PCR. FoxO1 gene expressions were highly in subcutaneous adipose and visceral adipose tissues, and higher in piglets than those in adults (P < 0.05). We showed that expression of endogenous FoxO1 in preadipocytes transfected with pBS/U6-siFoxO1-1748 expression vector was inhibited efficiently. After reducing expression of FoxO1, glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride (TG) content increased from day 1 to 9, and the time-course expressions of several key adipogenic genes mRNA, including peroxisome proliferator-activated receptor gamma on day 3, 5, and 7, adipocyte fatty acid binding protein on day 1, 3, and 5, and sirtuin1 on day 1, 3, and 5, were increased significantly (P < 0.05). Lipoprotein lipase was unrelevant to FoxO1. By using insulin-like growth factor-I treating, expression of FoxO1 reduced at day 3 and 5 (P < 0.05), and significant differentiation of porcine preadipocyte with increasing number of filled-lipid cell and size of lipid droplets, GPDH activity and TG content were promoted. These results suggested that porcine FoxO1 gene took part in the regulation of adipose and was a negative transcription regulation factor in preadipocyte differentiation.