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. Here, CHEK1 is linked to hepatocellular carcinoma.