Recent data suggested that endogenous hydrogen sulfide (H(2)S) contributes to the pathogenesis of diabetes. Here, we identified that cystathionine gamma lyase (CSE) was expressed in adipose tissue in rats and endogenously generated H(2)S. The CSE/H(2)S system exists in both rat adipocytes and pre-adipocytes. This system was up-regulated with aging, although a high level of glucose down-regulated the system in a concentration- and time-dependent manner. H(2)S inhibited the basal and insulin-stimulated glucose uptake of mature adipocytes, whereas administration of CSE inhibitors enhanced the glucose uptake of adipocytes. The PI3K but not K(ATP) channel pathway is involved in the inhibitory effect of H(2)S on glucose uptake. Finally, in fructose-induced diabetes in rats, we confirmed the up-regulated CSE/H(2)S system in adipose tissue, which was negatively correlated with glucose uptake in this tissue. Our findings suggest that H(2)S might be a novel insulin resistance regulator.