Piceamycin (<b>1</b>), a macrocyclic lactam isolated from the silkworm's gut (<i>Streptomyces</i> sp. SD53 strain), reportedly possesses antibacterial activity. However, the potential anticancer activity and molecular processes underlying <b>1</b> have yet to be reported. Colorectal cancer (CRC) is high-risk cancer and accounts for 10% of all cancer cases worldwide. The high prevalence of resistance to radiation or chemotherapy means that patients with advanced CRC have a poor prognosis, with high recurrence and metastasis potential. Therefore, the present study investigated the antitumor effect and underlying mechanisms of <b>1</b> in CRC cells. The growth-inhibiting effect of <b>1</b> in CRC cells was correlated with the upregulation of a tumor suppressor, N-myc downstream-regulated gene 1 (NDRG1). Additionally, <b>1</b> induced G<sub>0</sub>/G<sub>1</sub> cell cycle arrest and apoptosis and inhibited the migration of CRC cells. Notably, <b>1</b> disrupted the interaction between NDRG1 and c-Myc in CRC cells. In a mouse model with HCT116-implanted xenografts, the antitumor activity of <b>1</b> was confirmed by NDRG1 modulation. Overall, these findings show that <b>1</b> is a potential candidate for CRC treatment through regulation of NDGR1-mediated functionality.