The root of Platycodon grandiflorus (Jacq.) A. DC. ( P. grandiflorus ), Platycodonis Radix, has been commonly applied to prevent and treat human diseases including bronchitis, asthma and excessive phlegm. Platycodin D (PD), one of the most important therapeutic components of P. grandiflorus , has been reported to possess protective effect against alcohol and carbon tetrachloride induced hepatotoxicity. In this study, we examined the protective efficacy of PD on acetaminophen (APAP)-induced liver injury and possible underlying mechanisms in C57BL/6J mice. Administration of PD prior to APAP intoxication significantly ameliorated the increase in serum transferases, interleukin 1β (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), and hepatic malondialdehyde (MDA) and the depletion of glutathione (GSH) in mice. PD pretreatment decreased the expression of heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB) in presence of APAP. Moreover, PD treatment noticeably reduced APAP-induced hepatocyte necrosis and apoptosis evidenced by evaluating physiological and histological hepatocyte changes in mice. Finally, PD pretreatment significantly diminished c-Jun NH ₂ -terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38 phosphorylation induced by APAP. Collectively, PD pretreatment effectively protects hepatocytes against APAP-induced hepatotoxicity in mice through ameliorating oxidative stress, inflammatory response, and hepatocyte apoptosis.