Adiponectin is a polypeptide hormone that is secreted by adipocytes with insulin-sensitizing and anti-inflammatory properties. The current study was to further investigate the role of adiponectin on glucose uptake and its underlying mechanism by down-regulation of adiponectin in 3T3-L1 adipocytes. Transfection of short hairpin RNA (shRNA)-vector significantly decreased adiponectin mRNA expression and its protein level in the cells. The down-regulation of adiponectin markedly reduced the cellular glucose uptake rate and increased intracellular triglyceride content. To study the mechanism of the physiologic action of adiponectin, several key regulatory factors in insulin signaling pathway were examined. The mRNA expression of insulin receptor substrate (IRS)-1 in both basal and insulin-stimulated states were down-regulated in the transfected cells (72% and 52% of controls, respectively), and the insulin-stimulated IRS-1 tyrosine phosphorylation was also significantly decreased. Adiponectin-deficient cells showed marked down-regulations of peroxisome proliferator-activated receptor alpha, glucose transporter (GLUT)-1, GLUT-4, hormone-sensitive lipase (HSL), and adipose triglyceride lipase. These results thus demonstrated that transfection of shRNA-vector effectively reduced the expression of adiponectin in 3T3-L1 adipocytes accompanied with a significant decrease in cellular glucose uptake rate and an increase in intracellular triglyceride content. Our data also suggested that adiponectin deficiency impair insulin action in vitro probably through the IRS-1 pathway, and increase intracellular fat accumulation partially through HSL down-regulation.