Aims: The aim of this study was to evaluate the effects of Bifidobacterium lactis HY8101 on insulin resistance induced using tumour necrosis factor-alpha (TNF-alpha) in rat L6 skeletal muscle cells and on the KK-A(Y) mouse noninsulin-dependent diabetes mellitus (NIDDM) model. Methods and Results: The treatment using HY8101 improved the insulin-stimulated glucose uptake and translocation of GLUT4 via the insulin signalling pathways AKT and IRS-1(Tyr) in TNF-alpha-treated L6 cells. HY8101 increased the mRNA levels of GLUT4 and several insulin sensitivity-related genes (PPAR-c) in TNF-alpha-treated L6 cells. In KK-A(Y) mice, HY8101 decreased fasting insulin and blood glucose and significantly improved insulin tolerance. HY8101 improved diabetes-induced plasma total cholesterol and triglyceride (TG) levels and increased the muscle glycogen content. We observed concurrent transcriptional changes in the skeletal muscle tissue and the liver. In the skeletal muscle tissue, the glycogen synthesis-related gene pp-1 and GLUT4 were up-regulated in mice receiving HY8101 treatment. In the liver, the hepatic gluconeogenesis-regulated genes (PCK1 and G6PC) were down-regulated in mice receiving HY8101 treatment. Conclusions: Bifidobacterium lactis HY8101 can be used to moderate glucose metabolism, lipid metabolism and insulin sensitivity in mice and in cells. Significance and Impact of the Study: Bifidobacterium lactis HY8101 might have potential as a probiotic candidate for alleviating metabolic syndromes such as diabetes.