Synthesis of novel tetrakis(2,6-dimethyl-4-phenyl-1,4-dihydropyridinyl)methanes 5a–d by acid-catalyzed condensation of the tetrakis-aldehydes 6a–d with eight equivalents of 3-aminobut-2-enenitrile 2 is reported. The structures of 5a–d are confirmed by different spectral tools. In vitro, cytotoxic screening assay for novel tetrapodal 1,4-dihydropyridines (5a–d) was performed on five different human cell lines (HCT116, A549, MCF7, PC3, and HEPG2). The compounds showed higher cytotoxic activity against (A549, HCT116, and MCF7) cell lines. The loss of the cytotoxic activity was observed in the case of PC3 and HEPG2 cell lines. Compound 5b showed the highest cytotoxic activity against the three lines (A549, HCT116, and MCF7). In an attempt to know the mechanism followed by the compounds to inhibit cell proliferation, compound 5b was chosen for molecular studies. Compound 5b induced apoptotic inhibition of the proliferation of human colon adenocarcinoma HCT116 cells through induction of the tumor suppressor protein p53, BAX, and through the inhibition of anti-apoptotic proteins by decreasing BCL2 gene expression using real-time PCR. Regarding cell cycle analysis, compound 5b induced G1 arrest against the three lines (MCF7, HCT116, and A549). Compound 5b has been found to reduce apoptosis of human normal melanocytes HFB4 and normal fibroblasts BHK that has been treated with genotoxic factor H₂O₂. Moreover, compound 5b has a potent protective effect against DNA damage, as indicated by the in vitro studying of different concentrations of 5b against two different types of healthy DNA (calf-thymus DNA and pBR322 DNA).