OBJECTIVE Dietary factors and intestinal bacteria play an important role in the rapidly increasing incidence of obesity and its associated conditions, such as steatosis and insulin resistance. In the current study, we evaluated the effect of probiotics, and their mechanisms on diet-induced obesity, steatosis and insulin resistance. METHODS Wild-type male C57BL6 mice were fed either normal or high fat diets. Some mice received VSL#3 probiotics. Animal weight, hepatic steatosis, insulin resistance, and their relationship to hepatic Natural Killer T cells (NKT) cell number and inflammatory signaling were evaluated. RESULTS High fat diet induced a depletion of hepatic NKT cells thus leading to insulin resistance and steatosis. Oral probiotic treatment significantly improved the high fat diet-induced hepatic NKT cell depletion, insulin resistance and hepatic steatosis. This effect was NKT cell dependant, resulted from the attenuation of the tumor necrosis factor-alpha and IkappaB kinase inflammatory signaling, and led to an improved sensitivity in insulin signaling. CONCLUSIONS Probiotics improve high fat diet-induced steatosis and insulin resistance. These effects of probiotics are likely due to increased hepatic NKT cell numbers and reduced inflammatory signaling.