A unique feature of hepatitis B virus (HBV) infection in humans is that viral clearance heavily depends on the age of exposure. How-ever, the reason for this remains unclear. Here we show that gut microbiota contribute to the age dependence of HBV immunity in a hydrodynamic transfection mouse model. Although adult (12-wk-old) C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their young (6-wk-old) counterparts remained HBV-positive at 26 wpi. Ster-ilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented adult mice from rapidly clearing HBV. Young mice with the Toll-like-receptor (TLR) 4 mutation (C3H/HeJ) exhibited rapid HBV clearance. The results suggest that an immuno-tolerating path-way to HBV prevailed in young mice, before the establishment of gut bacteria, through a TLR4-dependent pathway and that the mat-uration of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance. liver tolerance | Toll-like 4 receptor | temporal-temperature gel electrophoresis | chronic hepatitis B | Kupffer cells