Autophagy plays a key role in supporting cell survival against chemotherapy-induced apoptosis. In this study, we found the chemotherapy agent SN-38 induced autophagy in colorectal cancer (CRC) cells. However, inhibition of autophagy using a small molecular inhibitor 3-methyladenine (3-MA) and ATG5 siRNA did not increase SN-38-induced cytotoxicity in CRC cells. Notably, another autophagy inhibitor chloroquine (CQ) synergistically enhanced the anti-tumor activity of SN-38 in CRC cells with wild type (WT) p53. Subsequently, we identified a potential mechanism of this cooperative interaction by showing that CQ and SN-38 acted together to trigger reactive oxygen species (ROS) burst, upregulate p53 expression, elicit the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). In addition, ROS induced by CQ plus SN-38 upregulated p53 levels by activating p38, conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death. Moreover, we showed induction of ROS and p53 by the two agents provoked the loss of LMP and ∆ψm. Altogether, all results suggested that CQ synergistically sensitized human CRC cells with WT p53 to SN-38 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 (a prodrug of SN-38) in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.