Cystathionine β-synthase (CBS) overexpression is related to the proliferation and migration of human colon cancers. Targeted therapy that inhibits CBS has achieved promising effects in colon cancer treatments, but no selective inhibitor of CBS is available. In our previous study, a natural biflavonoid compound, sikokianin C, was identified as a potent and selective inhibitor of CBS. However, the mode of action of this compound and its antitumor efficacy in vivo remain unknown. In the present study, we have demonstrated that sikokianin C selectively inhibits CBS activity in a competitive manner, and the five key residues involved in the binding of sikokianin C to the substrate channel of CBS protein were identified via a combination of molecular docking and site-directed mutagenesis. Additionally, we analyzed the antitumor efficacy of sikokianin C against human colon cancer cells in vitro and in vivo. Sikokianin C greatly suppressed the proliferation of HT29 colon cancer cells with an IC50 value of 1.6 μM, and CBS is the target of sikokianin C in mammalian cells, as evidenced by CBS knockdown analyses. Moreover, sikokianin C induced the apoptosis of HT29 cancer cells in a dose dependent manner. Treating mice with sikokianin C dramatically reduced the tumor volume and the weight of the colon cancer xenograft in vivo. These results indicate that the selective CBS inhibitor sikokianin C can potentially be used for the treatment of colon cancer.