In this study, two water-soluble pillar[5]arenes, [1-(3-(trimethyl-azane)propoxy)-4-methoxyhydro-chloridepillar[5]arene (asym-P5) and 1,4-bis(2-(trimethyl-azane)ethoxy)hydrobromidepillar[5]arene (sym-P5), were synthesized and characterized using fourier transform infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (1H-NMR and 13C-NMR). Cytotoxic and apoptotic effects of asym-P5 and sym-P5 on human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MDA-MB-231), human colon carcinoma (Caco-2), human prostate cancer (LnCap) and human embryonic kidney (HEK293) cell lines were determined by MTT, Annexin V-FITC apoptosis assays and quantitative analysis of mRNA levels of apoptosis related genes (Bax, Bcl-2, caspase 3, 8, and 9), respectively. asym-P5 not only reduced cell viability in all cell lines in a dose-dependent manner but also increased the apoptotic cell percentage significantly. sym-P5 was less effective than asym-P5 in inducing these effects in cancer cell lines. In RT-qPCR studies, it was observed that after asym-P5 treatment, Bax and caspase-3 mRNA levels increased, however, Bcl-2 levels decreased compared to the control group. As a result of the molecular docking, asym-P5 exhibited an energetically more favorable DNA binding affinity (-5.4 kcal/mol) than sym-P5 (-4.5 kcal/mol), where both ligands conformationally snug-fit into the major groove of DNA. These results demonstrated that asym-P5 had cytotoxic and apoptotic effects in cell lines.