Trapa bispinosa Roxb. is a traditional Chinese food which is well known for its medicinal properties. The shell of Trapa bispinosa has anticancer activity, maybe due to its high content of polyphenols. There are few studies on the chemical composition of Trapa bispinosa shells, then we isolated the active components from Trapa bispinosa shell and clarified the mechanism of its anticancer activity. One monomer compound was separated from the ethanol extract of the Trapa bispinosa shell by fractional extraction, silica gel, Sephadex LH-20 gel column chromatography and liquid phase separation. The structure, identified by NMR was 1,2,3,6-tetra-O-galloyl-β-D-glucose. The results of the CCK-8 assay showed that 1,2,3,6-tetra-O-galloyl-β-D-glucose could significantly inhibit the proliferation of gastric cancer SGC7901 cells, and the effect was close to that of 5-fluorouracil. Here, 1,2,3,6-tetra-O-galloyl-β-D-glucose could affect the cell cycle of SGC7901 cells. At the dose of 200 μg/mL and an incubation time of 48 h, SGC7901 cells remained in the G1 phase, apoptosis occurred, the intracellular calcium ion concentration increased and the mitochondrial membrane potential decreased. Transcriptome sequencing analysis showed that the differentially expressed genes were mainly enriched in the P53 signalling pathway associated with apoptosis. The results of qPCR and Western blot showed that 1,2,3,6-tetra-O-galloyl-β-D-glucose could induce apoptosis of SGC7901 cells by up-regulating the expression levels of P21, PUMA, PERP and IGF-BP3 genes, down-regulating the CyclinD gene, increasing the expression levels of cytochrome C, caspase-3, caspase-9 protein and decreasing that of the protein BCL-2.