Hepatotoxicity due to acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinic. Chlorogenic acid (CGA), a dietary polyphenol, was reported to prevent APAP-induced liver injury in our previous studies. This study aims to investigate the protection provided by CGA against APAP-induced hepatotoxicity via focusing on nuclear factor erythroid 2-related factor 2 (Nrf2) and extracellular regulated protein kinases (ERK)1/2. CGA prevented APAP-induced oxidative liver injury and enhanced Nrf2 activation in mice and in hepatocytes in vitro. CGA-provided the protection against APAP-induced hepatotoxicity was diminished after the application of Nrf2 siRNA in vitro and Nrf2 knockout mice in vivo. CGA enhanced the expression of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1), and their inhibitors reduced the protection provided by CGA against APAP-induced cytotoxicity in hepatocytes. Molecular docking results indicated the potential interaction of CGA with Nrf2 binding site in Kelch-like ECH-associating protein-1 (Keap1). CGA decreased the expression of protein phosphatases including PP2A subunit A (PP2A-A) and PP5, and induced the sustained ERK1/2 phosphorylation. Moreover, ERK1/2 inhibitors (U0126 and PD98059) and ERK2 siRNA abrogated CGA-induced Nrf2 phosphorylation and its subsequent transcriptional activation, and also reduced the protection provided by CGA against APAP-induced cytotoxicity in hepatocytes. These results suggest that CGA protects against APAP-induced hepatotoxicity by activating Nrf2 antioxidative signaling pathway via blocking the binding of Nrf2 to its inhibitor protein Keap1, and ERK1/2 plays a critical role in regulating CGA-induced Nrf2 transcriptional activation. CGA is a promising therapeutic agent for the detoxification of APAP-induced hepatotoxicity.