Colorectal cancer (CRC) is one of the most common malignancies worldwide. However, therapies against CRC have not been completely effective. Astragaloside IV (AS-IV) has shown anti-tumorigenic properties in certain cancers, but its role in CRC remains unclear. In this study, we investigated the therapeutic effect of AS-IV in CRC and explored its underlying mechanism. The results showed that AS-IV dose-dependently inhibited the proliferation of CRC cells and suppressed tumor growth in CRC xenograft mouse models. In addition, AS-IV promoted cell cycle arrest in the G0 phase, associated with increased expression of p21. Furthermore, flow cytometry demonstrated that AS-IV promoted apoptosis of CRC cells in a dose-dependent manner. AS-IV induced caspase-dependent apoptosis, which involved the increased release of cytochrome c and Omi from the mitochondria into the cytoplasm and the up-regulation of Bax/Bcl-2 ratio, as well as the activation of PARP and caspase cascade (caspase-3 and -9). Taken together, our study has identified a novel function of AS-IV and provided a molecular basis for AS-IV’s potential applications in the treatment of CRC and other cancers.