The present study was undertaken to validate the antitumor potential of Gymnosperma glutinosum from regional people's account, using the in vitro and in vivo L5178Y-R lymphoma murine model. Non-polar G. glutinosum crude extracts were tested on L5178Y-R cells. We found significant (p < 0.05) cytotoxic activity (up to 40%) of the hexane extract, which was further fractioned; fraction 1 (F1) was then observed to produce up to 51% apoptosis-mediated L5178Y-R cytotoxicity in vitro at concentrations lower than 0.98 microg/ml, and possess significant in vivo antitumor activity. This study may support further evaluation of active F1 in clinical trials.