Programmed cell death is a dynamic and critical mechanism of cell suicide in eukaryotes and prokaryotes. MazF is a ribonuclease protein involved in bacterial intracellular programmed death. This protein cleaves mRNAs at ACA sequences, leading to inhibition of protein synthesis and triggering cell death. Given that cancer is heterogenic and has varied susceptibility to treatment, we examined the impact of MazF proteins on the growth and viability of three cancer cell lines: MCF7, HT29, and AGS. These cell lines were transfected with ACA-less mazF mRNAs and evaluated for MazF-mediated cell death. The data illustrated that efficient MazF translation leads to a significant reduction in cell viability and is modulated by structural elements of ACA-less mazF mRNAs. In the presence of MazF, the levels of activated caspase-3 and -7 were significantly elevated in transfected cells, confirming the occurrence of apoptosis. We also quantified mRNA translation on a single-cell basis in MCF7 and AGS cell lines to examine MazF-mediated inhibition of protein synthesis. MazF expression significant decreases the levels of protein translation in the examined cell lines. This is the first report of MazF as a potential anti-cancer agent via induction of apoptosis in MCF7, AGS, and HT-29 cell lines.