Obesity has been identified as a major risk factor for type 2 diabetes, characterized by insulin resistance in insulin target tissues. Hypoxia-inducible factor 1α (HIF1α) regulates pathways in energy metabolism that become dysregulated in obesity. Earlier studies revealed HIF1α in adipose tissue is markedly elevated in hypoxic fat high-fat diet-fed mice that are obese and insulin resistant. Genetic ablation of HIF1α in adipose tissue decreased insulin resistance and obesity, accompanied by increased serum adiponectin level. However, the exact mechanism whereby HIF1α regulates adiponectin remains unclear. Here, acriflavine (ACF), an inhibitor of HIF1α, induced the expression of adiponectin and reduced the expression of Socs3 in cultured 3T3-L1 adipocytes. Mechanistic studies revealed that HIF1α suppressed the expression of adiponectin through a Socs3-Stat3 pathway. Socs3 was identified as a novel HIF1α target gene based on chromatin immunprecipitation and luciferase assays. Stat3 directly regulated adiponectin in vitro in cultured 3T3-L1 adipocytes. ACF was found to prevent diet-induced obesity and insulin resistance. In vivo, ACF also regulated the Socs3-Stat3-adiponectin pathway, and the inhibition of HIF1α in adipose tissue is essential for ACF to improve the Socs3-Stat3-adiponectin pathway to counteract insulin resistance. This study provides evidence for a novel target gene and signal transduction pathway in adipocytes, and that the inhibitors of HIF1α have potential utility for the treatment of obesity and type 2 diabetes.