BACKGROUND & AIMS: The costimulatory molecules OX40 and OX40L, members of the tumor necrosis factor (receptor) superfamily, play an important role in viral control through the activation of T cells. We speculate that activating the immune checkpoint OX40 may promote the inhibition of hepatitis B virus (HBV) replication. METHODS: To test this hypothesis, we investigated the expression dynamics of OX40/OX40L and studied the effects of activation of OX40 on HBV replication, and further explored the possible mechanism. RESULTS: We found that the percentage of T cells expressing OX40 was lower in adult patients with chronic hepatitis B (CHB) than in healthy adults and was negatively correlated with serum viral load. In contrast, the percentage of B cells and monocytes expressing OX40L was increased in adult patients with CHB and positively correlated with liver inflammatory indicators. The expression of OX40 in T cells and OX40L in monocytes was positively correlated with age in healthy donors. In addition, the levels of serum HBsAg and intrahepatic HBV DNA decreased in an HBV mouse model with an agonistic antibody that activates OX40. This viral inhibition process coincides with changes in liver inflammation, the ratio of T cell subsets, and T cell-related cytokines. Finally, we found that the OX40 activation-mediated inhibition of HBV replication was more dependent on CD8 CONCLUSIONS: The expression levels of the immune checkpoints OX40/OX40L in adult patients with CHB are closely related to virus clearance. The activation of OX40 can suppress HBV replication through a mechanism that is more dependent on CD8