© 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.Thallium (Tl+) is a heavy metal that causes toxicity in several organs, including the brain. Its cytotoxic profile, combined with its affinity for tumor cells when used as a radioligand for labeling these cells, suggests its potential use as antitumor therapy. In this study, glioblastoma cell lines C6 (from rat) and U373 (from human) were exposed to increased concentrations of thallium(I) acetate (5, 10, 50, 100, or 200 µM) and several toxic endpoints were evaluated, including loss of confluence and morphological changes, loss of cell viability, changes in cell cycle, and apoptosis. Tl+ was detected in cells exposed to thallium(I) acetate, demonstrating efficient uptake mechanism. Confluence in both cell lines decreased in a concentration-dependent manner (50–200 µM), while morphological changes (cell shrinkage and decreased cell volume) were more evident at exposures to higher Tl+ concentrations. For both parameters, the effects of Tl+ were more prominent in C6 cells compared to U373 cells. The same trend was observed for cell viability, with Tl+ affecting this parameter in C6 cells at low concentrations, whereas U373 cells showed greater resistance, with significant changes observed only at the higher concentrations. C6 and U373 cells treated with Tl+ also showed morphological characteristics corresponding to apoptosis. The cytotoxic effects of Tl+ were also assessed in neural and astrocytic primary cultures from the whole rat brain. Primary neural and astrocytic cultures were less sensitive than C6 and U373 cells, showing changes in cell viability at 50 and 100 µM concentrations, respectively. Cell cycle in both brain tumor cell lines was altered by Tl+ in G1/G2 and S phases. In addition, when combined with temozolamide (500 µM), Tl+ elicited cell cycle alterations, increasing SubG1 population. Combined, our novel results characterize and validate the cytotoxic and antiproliferative effects of Tl+ in glioblastoma cells.