--- - |2- Phoneutria nigriventer venom (PnV) decreases viability of brain and cervix tumor, but not of nontumor cells. PnV decreases the viability of glioma cells primarily by modulating the cell cycle, while the viability of glioblastoma cells is reduced mainly by inducing death. PnV delays migration of tumor cells and decreases invasiveness. The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U‐251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U‐251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules. - 'Journal of Cellular Physiology, Volume 234, Issue 2, Page 1398-1415, February 2019. '