Cepharanthine (CEP), an alkaloid derived from Stephania cepharantha Hayata, exhibits anti-cancer activities in various types of cancer. Colorectal cancer ranks as the third most common cancer worldwide and the third leading cause of cancer-related deaths among both men and women. Previous studies have reported that autophagic cell death serves as an alternative target for overcoming tumor resistance. This study aims to evaluate the anti-cancer effects of CEP on autophagy in colorectal cancer cells. The results showed that CEP significantly inhibited the growth of colorectal cancer cells (with an IC50 of 43.82 µM in HCT-116 cells and 12.08 µM in HT-29 cells at 24 hours). CEP induced autophagy by enhancing autophagic flux and the expression of autophagic-related proteins, including LC3-II, ATG5, and beclin-1. These changes correlate with an elevated expression of AMP-activated protein kinase (AMPK), an upstream activator of the autophagy pathway, in both cell lines. Our findings suggest that CEP could be a potential adjuvant chemotherapy treatment for colorectal cancer. These data support further in vivo investigations to establish the potential clinical applicability of CEP.