High-mobility group protein B1 (HMGB1) is a danger signaling molecule that has been found to trigger an effective antitumor immune response. However, the mechanisms underlying its antitumor effects are not fully understood. Here, we found that HMGB1 release induced by chemotherapy in patients with non–small cell lung cancer was negatively correlated with PD-1 expression on CD8+ T cells. In vitro analysis indicated that treatment with HMGB1 led to a significant decrease in the level of expression of PD-1 on CD8+ T cells. Further analysis demonstrated that HMGB1 reduced PD-1 expression by inducing dynamin-mediated internalization of the protein, leading to early endocytosis in the cytoplasm, and subsequently degradation in the lysosomes. In a xenograft model, HER2-targeted chimeric antigen receptor (CAR) T cells had enhanced function in the presence of HMGB1. These data identify a role for HMGB1 as a negative regulator of PD-1 signaling in lung cancer and the observed antitumor effect of HMGB1 on CAR T cells may provide a theoretical foundation for a new immunotherapy combination.