To examine the inhibitory activity of Free Polyphenol from Hull of Tartary Buckwheat (FPH) on HT-29 human colon cancer cells and the mechanisms of such activity, ultrasound-assisted acetone extraction was employed to obtain FPH. Polyphenol content was determined using the Folin-Ciocalteu colorimetric method, and FPH components were identified by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The inhibitory activity of FPH on HT-29 human colon cancer cells was evaluated using cell proliferation and colony formation assays as well as flow cytometry. FPH effects on Caspase-3 and Caspase-9 activity, and expression of apoptosis-related proteins Bax and Bcl-2 in HT-29 human colon cancer cells were detected using spectrophotometry and enzyme-linked immunosorbent assay (ELISA). Regulation of reactive oxygen species (ROS) production in HT-29 human colon cancer cells by FPH was analyzed using the DCFH-DA fluorescent probe method. FPH exhibited significant anti-proliferative activity against HT-29 cancer cells (IC50 value of 0.485 mg•mL-1), significantly reducing cancer cell colony survival, with a colony formation rate of only 4.62% at 500 μg•mL-1 FPH. FPH reduced expression of the proapoptotic factor Bax, inhibited the anti-apoptotic factor Bcl-2, and regulated Caspase-3 and Caspase-9 activities, thereby inducing tumor cell apoptosis and inhibiting their proliferation. The apoptosis rate reached 14.89% at 500 μg•mL-1 FPH. This study reveals application prospects for prevention of tumor cell proliferation and induction of apoptosis.