Damage-associated molecular patterns (DAMPs) have been implicated in sterile inflammation in various tissue injuries. High-mobility group box 1 (HMGB1) is a representative DAMP, and has been shown to transmit signals through receptors for advanced glycation end products (RAGEs) and Toll-like receptors (TLRs), including TLR2 and TLR4. HMGB1 does not, however, bind to TLRs with high affinity, therefore, the mechanism of HMGB1-mediated TLR activation remains unclear. In this study, we found that fluorescently labeled HMGB1 was efficiently internalized into macrophages through class A scavenger receptors. Although both M1 and M2 type macrophages internalized HMGB1, only M1 type macrophages secreted cytokines in response to HMGB1. The pan-class A scavenger receptor competitive inhibitor, maleylated-bovine serum albumin (M-BSA) inhibited HMGB1 internalization and reduced cytokine production from macrophages in response to HMGB1 but not to lipopolysaccharide (LPS). The C-terminal acidic domain of HMGB1 is responsible for scavenger receptor–mediated internalization and cytokine production. HMGB1 and TLR4 co-localized in macrophages, and this interaction was disrupted by M-BSA, suggesting that class A scavenger receptors function as co-receptors of HMGB1 for TLR activation. M-BSA ameliorated LPS-induced sepsis and DSS-induced colitis models in which HMGB1 has been shown to play progressive roles. These data suggest that scavenger receptors function as co-receptors along with TLRs for HMGB1 in M1-type inflammatory macrophages.