Holothurian glycosaminoglycan isolated from Apostichopus japonicus (named AHG) can suppress hepatic glucose production in insulin resistant hepatocytes, but its effects on glucose metabolism in vivo are unknown. The present study was conducted to investigate the effects of AHG on hyperglycemia in the liver of insulin resistant mice induced by a high-fat diet (HFD) for 12 weeks. The results demonstrated that AHG supplementation apparently reduced body weight, blood glucose level, and serum insulin content in a dose-dependent manner in HFD-fed mice. The protein levels and gene expression of gluconeogenesis rate-limiting enzymes G6Pase and PEPCK were remarkedly suppressed in the insulin resistant liver. In addition, although the total expression of IRS1, Akt, and AMPK in the insulin resistant liver was not affected by AHG supplementation, the phosphorylation of IRS1, Akt, and AMPK were clearly elevated by AHG treatment. These results suggest that AHG could be a promising natural marine product for the development of an antihyperglycemic agent.