Recently, a probiotic Lactobacillus rhamnosus GG (LGG) has shown several beneficial effects, including improved insulin sensitivity. To clarify the mechanism underlying the insulin-sensitizing effect of LGG, mice were orally administrated with LGG for 13 weeks, and their body weight, insulin sensitivity, and expression of genes related to glucose and lipid metabolism were examined. LGG-treated mice showed attenuated weight gain and enhanced insulin sensitivity in high fat diet group, while no change was observed in normal diet-fed group. The expression of fatty acid oxidative genes in the liver was increased and gluconeogenic genes were decreased. GLUT4 mRNA expression in skeletal muscle and adiponectin production in adipose tissue were significantly increased. This was corroborated with the increased activation of AMPK in skeletal muscle and adipose tissue. Taken together, these results indicate that LGG treatment improves insulin sensitivity and reduces lipid accumulation by stimulating adiponectin secretion and consequent activation of AMPK.