Probiotics are live microorganisms that confer a health benefit on the host, such as improvement of the intestinal environment, modulation of immune function and energy metabolism. Heat-killed probiotic strains have also been known to exhibit some physiological functions; however, the differences between live and heat-killed probiotics have not been well elucidated. In this study, we investigated the differences between live and heat-killed Bifidobacterium breve M-16V, a probiotic strain, in the regulation of immune function, intestinal metabolism and intestinal gene expression of the host using gnotobiotic mouse model and omics approaches. Both live and heat-killed cells of B. breve M-16V showed immune-modulating effects that suppressed pro-inflammatory cytokine production in spleen cells and affected intestinal metabolism; however, live cells exhibited a more remarkable effect in the regulation of intestinal metabolism and intestinal gene expression involved in nutrient metabolism. Our findings are valuable for considering the health benefits of live and heat-killed bacteria and the usefulness of different forms of probiotics.