Isaindigotone is an alkaloid containing a pyrrolo-[]quinazoline moiety conjugated with a benzylidene group and isolated from the root of Fort. However, further anticancer activities of this alkaloid and its derivatives have not been fully explored. In this work, a novel isaindigotone derivative was synthesized and three different gastric cell lines and one human epithelial gastric cell line were used to study the anti-proliferation effects of the novel isaindigotone derivative BLG26. HGC27 cells and AGS cells were used to further explore the potential mechanisms. BLG26 exhibited better anti-proliferation activities in AGS cells with a half-maximal inhibitory concentration (IC) of 1.45 μM. BLG26 caused mitochondrial membrane potential loss and induced apoptosis in both HGC27 cells and AGS cells by suppressing mitochondrial apoptotic pathway and PI3K/AKT/mTOR axis. Acute toxicity experiment showed that LD (median lethal dose) of BLG26 was above 1000.0 mg/kg. This research suggested that BLG26 can be a potential candidate for the treatment of gastric cancer.