Several studies revealed that gut microbiota affects the hepatic drug-metabolizing enzyme cytochrome P450 (Cyp). We hypothesized that individual gut microbiota variations could contribute to CYP activity. Human flora-associated (HFA) mice are established from germ-free mice using human feces and are often used to determine the effect of the human gut microbiota on the host. This study generated two groups of HFA mice using feces from two healthy individuals. Then, the composition of gut microbiota and hepatic Cyp activity was compared to analyze the effects of gut microbiota in healthy individuals on hepatic Cyp activity. A principal coordinate analysis based on the UniFrac distance for the composition of the cecal and fecal microbiota revealed apparent differences between the recipient groups. Hepatic Cyp, which is a marked difference in Cyp3a activity and Cyp3a11 gene expression, was observed between the recipient groups. Cyp2c and Cyp1a activities did not differ between recipient groups, with significantly lower enzymatic activities in recipients than in germ-free mice. These results indicate that the human gut microbiota affects hepatic Cyp activity. Especially, human gut microbiota composition differences have a pronounced effect on Cyp3a activity via Cyp3a11 gene expression regulation. Therefore, human gut microbiota variations among individuals may affect numerous drug metabolism, leading to drug efficacy and toxicity.