In conclusion, we demonstrated that Mus81 knockdown could significantly improve the chemosensitivity of HCC cells in vitro and in vivo by inducing S‐phase arrest and promoting apoptosis through CHK1 activation, which in turn activates CHK1/CDC25A/CDK2 pathway joined by CHK1/p53/Bax/Caspase‐3 branch. This evidence concerns the gene TP53 and hepatocellular carcinoma.