Colorectal cancer (CRC) is the third commonly diagnosed malignancy and the second leading cause of cancer death worldwide. Development of novel chemotherapeutics is crucial. Natural products are the main source of drug discovery, and epipolythiodioxopiperazine (ETP) alkaloids are one kind of them have been reported to have potent biological activities. In the present study, we first isolated Chaetocochin J (CJ), an ETP alkaloid from the secondary metabolites of Chaetomium sp, and studied the anti-CRC activity and mechanism of it. The results showed that CJ exhibits potent proliferation inhibition effect, its IC to CRC cells are around 0.5 µM. CJ also induces apoptosis of CRC cells in a dose-dependent manner, and this effect is stronger than topotecan. In addition, CJ treatment triggers autophagic flux in CRC cells, inhibition of autophagy by chloroquine didn’t affect CJ-induced apoptosis and growth inhibition, suggesting CJ may simultaneously induced apoptosis and autophagy in CRC cells. We further explored the mechanism of action, and found that CJ exerts its anti-CRC function via AMPK and PI3K/AKT/mTOR pathways and further regulation of their downstream signaling cascade in CRC cells, including apoptosis and autophagy. These data potently suggest that CJ may be a potential drug candidate for CRC treatment.