Swertia petiolata is an important medicinal herb and has been shown to possess tremendous pharmacological potential. It belongs to family Gentianaceae consisting of about 1600 species. The objective of the present was to evaluate cytotoxic effect of methanolic extract of S. petiolata against a panel of different cancer cell lines. Our results revealed that the extract exhibited strong cytotoxic activity against gastric cancer cell line SNU-5 with an IC50 value of 64μg/ml. Our results showed that the extract decreased the viable SNU-5 cells and also reduced the clonogenic potential in a concentration dependent manner. The extract induced cell death through apoptosis as was evident from Annexin V/PI and DAPI staining. S. petiolata methanolic extract also promoted the release of reactive oxygen species (ROS) and decreased the levels of mitochondria membrane potential providing a strong biochemical evidence for apoptosis. To gain insights about the composition of the extract, LC–MS analysis was carried and the major constituents were identified. The results indicated the major constituents were mostly phenolics and flavonoids. The characteristic compounds (swerchirin and genistein) of genus Swertia and family Gentianaceae were also tentatively identified and may be one of the reasons for the cytotoxic activity of the extract. We therefore propose that the cytotoxic activity of S. petiolata extract is due to induction of apoptosis through generation of ROS.