p53 is a tumor suppressor with a pro-death role in many conditions. However, in some contexts, evidence supports a pro-survival function. p53 has been shown to be activated in acetaminophen (APAP) toxicity but the impact of this on toxicity is uncertain. In the present study, we have found that p53 plays a protective role in APAP-induced liver injury. We inhibited p53 using three different approaches in mice, pifithrin-α (PFTα), knockdown of p53 expression with antisense oligonucleotide, and p53 knockout. Mice were treated with APAP (300 mg/kg) i.p. and after 24 h in all three conditions, the liver injury was more severe as reflected in higher ALT levels and great area of necrosis in histology of the liver. Conversely, a p53 activator, nutlin-3a, decreased the liver injury induced by APAP. In the p53 inhibition models, enhanced sustained JNK activation was seen in the early time course, while the JNK was suppressed with the p53 activator. In conclusion, p53 plays a novel protective role in APAP induced liver injury through inhibiting the activation of JNK, a key mediator in APAP-induced oxidative stress.