Hepatic ischemia‐reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR‐induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll‐interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal‐regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor–associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N‐terminal dimerization and the subsequent activation of downstream mitogen‐activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip‐regulated ASK1–MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N‐terminal dimerization and the resultant c‐Jun N‐terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.