Update contact name. Update contact information. Update publication information. Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options. There is an urgent need for developing novel therapeutic agents against CRC with good treatment efficiency and low toxicity. Odoroside A (OA) is an active compound isolated from the leaves of Nerium oleander Linn. (Apocynaceae). This study aims to examine the anticancer bioactivity of OA against CRC cells and to investigate the action mechanisms involved. As a result, OA was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any significant cytotoxicity in normal colorectal epithelial cells. Mechanistically, quantitative proteomics coupled with bioinformatics was performed to explore the OA-regulated proteins and signaling pathways. As suggested by the proteomic analysis, Western blot analysis and flow cytometry validated that exposure of CRC cells to OA caused cell cycle arrest and apoptosis, accompanied with the activation of the ROS/p53 signaling pathway. This observation demonstrated that OA, as a natural product, can induce ROS to suppress tumor cell growth, implicating a novel therapeutic agent against CRC without obvious side effects.