Studies in cervical and prostate cancer cells have reported that frequent consumption of foods rich in glycine betaine (GB) and choline have beneficial effects against some types of cancer. However, the role of GB against the human colorectal adenocarcinoma cell line HT-29 has not yet been elucidated. Therefore, this study aimed to evaluate the effect of GB on p53 and caspase-3 expression, which regulate cellular processes such as cell proliferation and apoptosis, respectively, on HT-29 cells. HT-29 cells were treated with GB at 5 mg/mL, 15.6 mg/mL, 31.2 mg/mL, and 62.5 mg/mL, after which RNA purification and cDNA synthesis were performed, followed by qPCR to detect the relative expression of p53 and caspase-3, using GAPDH as a reference gene, and protein levels were determined by ELISA. Results indicated that in HT-29 cells treated with GB at 62.5 mg/mL, the protein levels of p53 significantly (p < 0.05) increased to 45 U/mL, as compared with cells without GB (21 U/mL), whereas caspase-3 increased to 30 ng/mL, as compared with control cells (20.13 ng/mL). Therefore, we conclude that GB at high concentrations decreases cell proliferation and induces apoptosis in HT-29 cells.