Calorie restriction has been regarded as the only experimental regimen that can effectively lengthen lifespan in various animal models, but the actual mechanism remains controversial. The gut microbiota has been shown to have a pivotal role in host health, and its structure is mostly shaped by diet. Here we show that life-long calorie restriction on both high-fat or low-fat diet, but not voluntary exercise, significantly changes the overall structure of the gut microbiota of C57BL/6 J mice. Calorie restriction enriches phylotypes positively correlated with lifespan, for example, the genus Lactobacillus on low-fat diet, and reduces phylotypes negatively correlated with lifespan. These calorie restriction-induced changes in the gut microbiota are concomitant with significantly reduced serum levels of lipopolysaccharide-binding protein, suggesting that animals under calorie restriction can establish a structurally balanced architecture of gut microbiota that may exert a health benefit to the host via reduction of antigen load from the gut.