Cytolysin A (ClyA), a pore-forming toxin, exhibits cytotoxicity in various cell types, but epithelial cells often display resistance. This study investigates ClyA’s effects on five colon cancer epithelial cell lines (HCT8, DLD1, H4, HCT116, Caco-2), revealing cell line–specific responses. Cells treated with increasing ClyA concentrations (up to 1000 nM) for 48 hours were assessed for viability and membrane integrity using propidium iodide (PI) and Hoechst 33342 staining, imaged via sparkCyto, and analyzed with Cellpose. HCT8, DLD1, and H4 cells showed significant cytotoxicity, with increased PI-positive cells and a concentration-dependent viability decline (MTS assay), while HCT116 displayed intermediate sensitivity at 1000 nM, and Caco-2 remained resistant. Morphological analysis using a holomonitor revealed reduced cell area and thickness in HCT8 cells, but not in HCT116. Flow cytometry with Annexin V/PI staining in HCT116 cells confirmed ClyA induces primarily necrotic cell death, with minimal apoptosis (P ≤ 0.01). In 3D spheroid models of DLD1 and HCT116 cells, ClyA caused a concentration-dependent increase in PI fluorescence and reduced spheroid core area, with DLD1 showing greater sensitivity. ClyA’s pore-forming activity was validated using liposomes derived from cellular lipid extracts (ECLE) and giant plasma membrane-derived vesicles (GPMVs). Negative staining of ECLE liposomes revealed ring-like pore assemblies, while GPMV assays with FITC-dextran confirmed membrane permeabilization. ClyA internalization in HCT116 cells was studied using pathway-specific inhibitors and flow cytometry, showing primary uptake via clathrin- and caveolin-mediated endocytosis (inhibited by dynasore, chlorpromazine, methyl-β-cyclodextrin), with a minor role for PI3K-dependent macropinocytosis (inhibited by LY294002). Live-cell confocal microscopy further revealed ClyA co-localization with early endosomes in HCT8 cells. Cytotoxicity assays in HCT8 and DLD1 cells under varying pH conditions demonstrated enhanced ClyA toxicity at lower pH (e.g., 50% cytotoxicity at pH 6.8 after 4 hours in DLD1 cells, versus none at pH 7.4), confirmed by increased necrotic and late apoptotic populations at pH 6.8 (Annexin V/PI staining). These findings highlight ClyA’s cell line–specific cytotoxicity, necrotic mechanism, pore-forming activity, endocytic uptake, and pH-dependent effects, providing insights into its potential role in colon cancer cell responses.