Gut microbiota alternations are associated with various disorders. In this study, gut microbiota changes were investigated in a methionine-choline deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) rodent model, and the effects of administering Lactobacillus casei strain Shirota (LcS) on the development of NASH were also investigated. Mice were divided into three groups, given the normal chow diet (NCD), MCD diet, or the MCD diet plus daily oral administration of LcS for 6 weeks. Gut microbiota analyses for the three groups revealed that lactic acid bacteria such as Bifidobacterium and Lactobacillus in feces were markedly reduced by the MCD diet. Interestingly, oral administration of LcS to MCD diet-fed mice increased not only the L.casei subgroup but also other lactic acid bacteria. Subsequently, NASH development was evaluated based on hepatic histochemical findings, serum parameters and various mRNA and/or protein expression levels. LcS intervention markedly suppressed MCD-diet induced NASH development, with reduced serum lipopolysaccharide concentrations, suppression of inflammation and fibrosis in the liver, and reduced colon inflammation. Therefore, reduced populations of lactic acid bacteria in the colon may be involved in the pathogenesis of MCD diet-induced NASH, suggesting normalization of gut microbiota to be effective for treating NASH.