As part of the screening program for anticancer agents from natural sources, the sesquiterpene lactone goyazensolide (GZL) was identified as a potent NF-κB inhibitor. The hollow-fiber assay was used to evaluate the anti-tumor efficacy of GZL in vivo. The mechanistic effects of GZL were evaluated in the HT-29 colonic cell line to reveal the pathway through which GZL exerts its effects. NF-κB subunits p65 and p50 were inhibited, and the upstream mediator IκB kinase (IKKβ) was downregulated in a dose-dependent manner. Apoptosis was mediated by caspase-3, and cell cycle arrest was detected in G1-phase. Consequently, 96% of the cell population was in sub G1-phase after treatment with GZL (10 μM).The antitumor effect of GZL was observed at a dose of 12.5 mg/kg. Cell adhesion was affected as a result of NF-κB inhibition. GZL appears to selectively target the transcription factor NF-κB. In summary, GZL sensitizes HT-29 colon cancer cells to apoptosis and cell death in a dose-dependent manner both in vivo and in vitro, through NF-κB inhibition (IC50 = 3.8 μM). Thus, it is a new potent lead compound for further development into a new effective chemotherapeutic agent.