Resistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELM were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELM on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELM overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELM transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELM, suggesting the insulin resistance-inducing effect of RELM to be direct. Furthermore, it was shown that RELM acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELM transgenic mice. In conclusion, RELM, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELM may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELM is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents.