Liver fibrosis is a worldwide clinical issue. The activation of hepatic stellate cells (HSCs) is the central event during the hepatic fibrotic response. However, the exact mechanisms related to HSC activation and the connection between hepatocytes and HSCs remain unclear. We elucidated the mechanism by which the nuclear-damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1) was released from the impaired hepatocytes and induced endoplasmic reticulum stress to activate HSCs. In this work, we demonstrated that HMGB1 can be released from hepatocytes and the released HMGB1 activates the HSCs via ER stress at the transcriptional level which was dependent on the activation of both the TLR4 and RAGE signaling pathways rather than the TLR2 signaling pathway. HMGB1 induction of proinflammatory cytokines interleukin (IL)-1 beta and IL-18 release was dependent on ER stress. In vivo, stable inhibition of HMGB1 suppressed liver fibrosis. These results suggest that under damage condition, HMGB1 can be secreted from injured hepatocytes and activates TLR4- and RAGE signaling pathways to induce ER stress which activates HSCs. Moreover, HMGB1 can produce multiple inflammatory mediators through ER stress, which, in turn, promote liver fibrosis.