Quinacrine (QNC), antiprotozoan drug commonly used against Malaria and Giardiasis , has been recently tried for rheumatics and prion diseases via drug repositioning. In addition, several reports suggest antitumor effects of QNC through suppression of NF-B and activation of p53. This study demonstrates the anticancer effect of QNC via a novel pathway through the elimination of checkpoint kinase 1/2 (Chk1/2) under p53-inactivated conditions. Inhibition of p53 by PFT-α or siRNA promotes QNC-induced apoptosis in normal fibroblast and p53-intact cancer cells. Considering that Chk1/2 kinases exert an essential role in the control of cell cycle, inhibition of Chk1/2 by QNC may induce cell death via uncontrolled cell cycle progression. Indeed, QNC reduces Chk1/2 expression under p53-impaired cancer cells and induces cell death in the G 2 –M phase. QNC increases the binding between p-Chk1/2 and β-TrCP and promotes proteasome-dependent degradation. Moreover, QNC treatment displayed antitumor effects in a Villin-Cre;p53 +/LSL-R172H intestinal cancer mouse model system as well as HCT116 p53 –/– xenografts. Implications: QNC has been used for the past over 70 years without obvious side effects, as such it is a plausible drug candidate for relapsed cancers, small-cell lung cancer, breast cancer as well as various p53-inactivated human malignancies. Mol Cancer Res; 16(6); 935–46. ©2018 AACR .