Accidental or intentional misuse of acetaminophen (APAP) is the leading cause of acute liver failure in the Western world. Although mechanisms that trigger APAP-induced liver injury (AILI) are well known, those that halt the progression of APAP liver disease and facilitate liver recovery are less understood. Heparan sulfate proteoglycans (HSPGs) bind to and regulate various tissue injury factors through their heparan sulfate (HS) chains, but the importance of HSPGs in liver injury in vivo remains unknown. Here, we examined the role of syndecan-1, the major cell-surface HSPG of hepatocytes, in AILI. Ablation of syndecan-1 in mice led to unopposed progression of liver injury upon APAP overdose. However, direct APAP hepatoxicity and liver injury at early times post-APAP overdose were unaffected by syndecan-1, suggesting that syndecan-1 influences later mechanisms that lead to liver repair. The exuberant liver injury phenotypes in syndecan-1 null (Sdc1^−/−) mice were traced to a deficiency in protein kinase B (Akt) activation in hepatocytes, which led to a delayed increase in glycogen synthase kinase-3β (GSK-3β)-mediated hepatocyte apoptosis. Inhibition of Akt worsened, whereas inhibition of GSK-3β and caspases protected mice from AILI. Moreover, administration of purified syndecan-1, HS, or engineered heparan compounds containing 2-O-sulfate groups rescued Sdc1^−/− mice from AILI by potentiating Akt signaling and inhibiting GSK-3β-mediated apoptosis in hepatocytes. In addition, HS showed a significantly prolonged therapeutic efficacy as compared to N-acetylcysteine. Conclusion: These results demonstrate that 2-O-sulfated domains in syndecan-1 HS halt disease progression and promote liver repair by enhancing hepatocyte survival in AILI. We propose that syndecan-1 is a critical endogenous factor that controls the balance between prosurvival signaling and apoptosis in hepatocytes in APAP liver disease. (Hepatology 2017;66:1601–1615).