Due to its extended latency and high prevalence, human colorectal carcinoma (hCRC) represents an attractive candidate for dietary supplements that have shown remarkable success in tumor treatment. One natural dietary flavonoid, Kaempferol, exhibits numerous pharmacological activities, including antioxidant, antimicrobial, antidiabetic, and anticancer effects. This study aimed to investigate the anti-proliferative effects of Kaempferol and clarify its molecular mechanism on hCRC. High-pressure liquid chromatography (HPLC) and UV–Vis spectroscopy confirmed that the molecular structure of Kaempferol remained stable for up to 48 h. Confocal microscopy further demonstrated that Kaempferol localized in the cytoplasm of cells. The anticancer properties of Kaempferol were evaluated against several human cancer cell lines, including DLD-1 (colorectal carcinoma), A549 (lung cancer), HUH-7 (hepatocarcinoma), HeLa (cervical cancer), and NCI-h295R (adrenal gland cancer), as well as the healthy epithelial cell line CCD-18Co. Kaempferol significantly inhibited the proliferation of DLD-1 cells, with an IC<inf>50</inf> value of 49.55 µM. Kaempferol treatment increased endoplasmic reticulum (ER) stress and apoptosis by 47% (p < 0.001). Kaempferol also modulated the mRNA and protein expression of BcL-2, Bax, Caspase-3, Caspase-9, Caspase-12, Nf-κB, p53, adenomatous polyposis coli (APC), MLH-1, Smad-4, XBP-1, PERK, and ATF6. Molecular docking and 100 ns molecular dynamics simulations revealed a stable binding conformation of Kaempferol within the BcL-2 active site, supported by key hydrogen bonds and hydrophobic interactions with residues such as Asn140 and Tyr199. MM/GBSA free energy and ligand efficiency calculations further confirmed its favorable binding characteristics. Collectively, these findings demonstrate that Kaempferol induces colorectal cancer cell death through ER stress–mediated apoptosis and BcL-2 modulation, positioning it as a promising candidate for further preclinical development.