Among digestive organs, the liver and the large intestine are abundant in T cells expressing NK1.1. NK1.1+ T cells in the liver are mostly CD1d-dependent whereas those in the large intestine are CD1d-independent. In this study, we investigated the effects of Lactobacilli on NK1.1+ T cells in the digestive organs of mice. C57BL/6 mice were orally given a dietary supplement prepared from mixed cultures of eight strains of Lactobacilli. Oral administration of Lactobacilli to mice resulted in the selective expansion of NK1.1+ T cells in the large intestine. These colon NK1.1+ T cells activated by Lactobacilli were found to express IFN-gamma mRNA. The level of IFN-gamma in the serum was also elevated by the administration of Lactobacilli. Our results suggest that Lactobacilli selectively activate CD1d-independent NK1.1+ T cells in the large intestine to produce IFN-gamma and therefore modulate Th1 immune responses.