Urotensin-II (U-II) is an endogenous peptide agonist of a G protein-coupled receptor-urotensin receptor. There are many conflicting findings about the effects of U-II on blood glucose. This study aims to explore the effects of U-II on glucose metabolism in high-fat diet-fed mice. Male C57BL/6J mice were fed a 45% high-fat diet or chow diet and were administered U-II intraperitoneally for in vivo study. Skeletal muscle C2C12 cells were used to determine the effects of U-II on glucose and fatty acid metabolism as well as mitochondrial respiratory function. In this study, we found that chronic U-II administration (more than 7 days) ameliorated glucose tolerance in high-fat diet-fed mice. In addition, chronic U-II administration reduced the weight gain and the adipose tissue weight, including visceral, subcutaneous, and brown adipose tissue, without a significant change in blood lipid levels. These were accompanied by the increased mRNA expression of the mitochondrial thermogenesis gene Ucp3 in skeletal muscle. Furthermore, in vitro treatment with U-II directly enhanced glucose and free fatty acid consumption in C2C12 cells with increased aerobic respiration. Taken together, chronic U-II stimulation leads to improvement on glucose tolerance in high-fat diet-fed mice and this effect maybe closely related to the reduction in adipose tissue weights and enhancement on energy substrate utilization in skeletal muscle.