Background and Purpose: Colorectal cancer (CRC) is one of the most diagnosed cancer worldwide, and effective chemotherapeutic drugs for CRC still remain a challenge. Ziyuglycoside II (Ziyu II) is one of the major active compounds of Sanguisorba officinalis L. Previous study has identified Ziyu II as a potent anti-tumor agent, but limited data on the efficacy and potential mechanism of Ziyu II in the treatment of CRC. Experimental Approach: CRC cells were used to examine the tumor suppression effect of Ziyu II alone or in combination with autophagy inhibitors in vitro and in vivo. A variety of biochemical assays were conducted to elucidate the underlying mechanisms of Ziyu II in CRC cells. Key Results: Ziyu II exhibits antitumor activity against CRC cells both in vitro and in vivo. It demonstrated that treatment with Ziyu II induced apoptosis via accumulation of reactive oxygen species (ROS). Intriguingly, Ziyu II treatment triggered complete autophagic flux in CRC cells. Inhibition of autophagy partially reversed Ziyu II-induced growth inhibition in CRC cells, suggesting a cytotoxic role of Ziyu II-induced autophagy. Mechanistic studies showed that Ziyu II induced autophagy by inhibiting Akt/mTOR pathway, and Akt reactivation partially reduced Ziyu II-induced autophagy. Notably, Ziyu II improves the sensitivity of CRC cells to the first-line chemotherapeutic drugs 5-fluorouracil. Conclusion and Implications:This study provides new insights into the molecular mechanisms of Ziyu II-mediated CRC suppression involving induction of both apoptosis and autophagy, and establishes potential applications of Ziyu II for clinical CRC treatment.