Our previous study revealed that microRNA-125a-5p plays a crucial role in regulating hepatic glycolipid metabolism by targeting STAT3 in type 2 diabetes mellitus (T2DM). Dioscin, a major active ingredient in Dioscoreae nipponicae rhizomes, displays various pharmacological activities, but its role in T2DM has not been reported. The aim of this study was to investigate the effect of dioscin on T2DM and elucidate its potential mechanism. The effect of dioscin on glycolipid metabolic disorder in insulin-induced HepG2 cells, palmitic acid-induced AML12 cells, high-fat diet- and streptozotocin- induced T2DM rats, and spontaneous T2DM KK-Ay mice were evaluated. Then, the possible mechanisms of dioscin were comprehensively evaluated. Dioscin markedly alleviated the dysregulation of glycolipid metabolism in T2DM by reducing hyperglycemia and hyperlipidemia, improving insulin resistance, increasing hepatic glycogen content, and attenuating lipid accumulation. When the mechanism was investigated, dioscin was found to markedly elevate miR-125a-5p level and decrease STAT3 expression. Consequently, dioscin increased phosphorylation levels of STAT3, PI3K, AKT, GSK-3β, and FoxO1 and decreased gene levels of PEPCK, G6Pase, SREBP-1c, FAS, ACC, and SCD1, leading to an increase in glycogen synthesis and a decrease in gluconeogenesis and lipogenesis. The effects of dioscin on regulating miR-125a-5p/STAT3 pathway were verified by miR-125a-5p overexpression and STAT3 overexpression. Dioscin showed potent anti-T2DM activity by improving the inhibitory effect of miR-125a-5p on STAT3 signaling to alleviate glycolipid metabolic disorder of T2DM.