Our previous study demonstrated that pre-administration of 1O, 20O-diacetyl kamebakaurin (Ac2KA) protected against acetaminophen (APAP)-induced hepatotoxicity. In the current study, we aimed to investigate whether post-administration of Ac2KA also protects against APAP-induced hepatotoxicity. Eight-week-old male C57BL/6J mice were fasted and then intraperitoneally injected with 450 mg/kg APAP or saline. At 60-min after the APAP injection, Ac2KA (50 mg/kg) or an ethanol/olive oil emulsion was orally administered. At 16-hr after the injection, the mice were killed, and blood samples were collected for plasma analysis. As a positive control, we used N-acetylcysteine (200 mg/kg, i.p.). Post-treatment with Ac2KA significantly attenuated APAP-induced plasma alanine aminotransferase and aspartate aminotransferase levels. Ac2KA administration also decreased the APAP-induced hepatic malondialdehyde concentration. Moreover, histological evaluation supported these observations. Our results show that Ac2KA exerts protective effects against APAP-induced hepatotoxicity when administered as both pretreatment and post-treatment.