A series of squaramates and squaramides have been synthesized and their cytotoxic activity has been investigated in different cancer cell lines. Among the studied compounds, squaramide 34 showed a potent and selective cytotoxicity against the human gastric cancer cell line HGC-27. Studies directed to elucidate the mechanism of induced cell death were performed. Cell cycle distribution analysis and cell death studies showed that compound 34 induces cell cycle arrest at the G1 phase and caspase-dependent apoptosis. In conclusion, squaramide 34 can be considered a potential anticancer agent for gastric carcinoma.