Acetaminophen(APAP)overdose is the leading cause of drug-induced liver injury,and its prognosis depends on the balance between hepatocyte death and regeneration.Sirtuin 6(SIRT6)has been reported to protect against oxidative stress-associated DNA damage.But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear.In this study,the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment,respectively.Sirt6 knockdown in AML 12 cells aggravated APAP-induced hepatocyte death and oxidative stress,inhibited cell viability and proliferation,and downregulated CCNA1,CCND1 and CKD4 protein levels.Sirt6 knockdown signifi-cantly prevented APAP-induced NRF2 activation,reduced the transcriptional activities of GSTμ and NQO1 and the mRNA levels of Nrf2,Ho-1,Gsta and Gstfi.Furthermore,SIRT6 showed potential protein interaction with NRF2 as evidenced by co-immunoprecipitation(Co-IP)assay.Additionally,the protec-tive effect of P53 against APAP-induced hepatocytes injury was Sirt6-dependent.The Sirt6 mRNA was significantly down-regulated in P53-/mice.P53 activated the transcriptional activity of SIRT6 and ex-erted interaction with SIRT6.Our results demonstrate that SIRT6 protects against APAP hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation,and provide new insights in the function of SIRT6 as a crucial docking molecule linking P53 and NRF2.