Background and Purpose: Colorectal cancer (CRC) is an exceptionally deadly disease, whereas therapeutic drugs for CRC have presented a serious shortage over the past few decades. Natural products have become an inexhaustible source of anticancer drugs. Natural alkaloid N-hydroxyapiosporamide (NHAP) exerts antitumor effects, but its effect and mechanism in CRC remain unclear. This study aimed to reveal the antitumor target of NHAP and identify NHAP as a promising leading compound for CRC. Experimental Approach: The inhibitory effects of NHAP on growth of CRC cells were determined by SRB and colony formation assays. Various biochemical methods were used to investigate molecular mechanisms of action for NHAP, including western blotting, RT-PCR, flow cytometry, immunofluorescent staining, cell transfection and luciferase assay, RNA-seq analysis, ELISA and immunoprecipitation analysis. Mouse endotoxin shock model, CRC xenograft model and azoxymethane model were used to assess the in vivo anti-tumor effect of NHAP against CRC. Key Results: NHAP exhibited potent cytotoxicity, induced both apoptosis and autophagic cell death of CRC cells, and inhibited the NF-κB signaling pathway by blocking the interaction of TAK1-TRAF6 proteins. NHAP also markedly inhibited CRC tumour growth in vivo without obvious toxicity and possessed superior pharmacokinetic characteristics. Conclusion and Implications: These findings identify, for the first time, that natural alkaloid NHAP is a novel NF-κB inhibitor with potent anti-tumor activity against CRC in vitro and in vivo. This study clarifies the anti-tumor target of NHAP against CRC, which will contribute to the future development of NHAP as a novel therapeutic leading compound for CRC.