Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring ‘regenerative’ therapies. Therefore, the role of the Wnt/β-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. β-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of β-catenin for 1 to 12 hours. These data were also verified by increased expression of the β-catenin surrogate target glutamine synthetase. β-Catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3β and an increase in levels of casein kinase 2α, and led to increased cyclin-D1, another known β-catenin target. These observations were next substantiated in β-catenin conditional-null mice (β-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in β-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in β-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear β-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of β-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.