In this study, we attempt to target Arginine auxotrophy in glioblastoma multiforme (GBM) cells using a pegylated recombinant human Arginase I cobalt [HuArgI (Co)-PEG5000]. We tested and characterized the activity of HuArgI (Co)-PEG5000 on a panel of 9 GBM cell lines and on human fetal glial cells (SVG-p12). HuArgI (Co)-PEG5000 was cytotoxic to all GBM cells tested. SVG-p12 cells were not sensitive demonstrating the selective cytotoxicity of HuArgI (Co)-PEG5000-induced arginine deprivation. Addition of L-citrulline led to the rescue of 6 GBM cell lines but only at concentrations of 11.4 mM, reflecting the extent of arginine auxotrophy in GBM. The ability of L-citrulline to rescue cells was dependent on the expression of argininosuccinate synthetase-1 (ASS1) with the cells that were not rescued by L-citrulline being negative for ASS1 expression. Knocking-down ASS1 reversed the ability of L-citrulline to rescue GBM cells, further illustrating the dependence of arginine auxotrophy on ASS1 expression. Inhibition of autophagy increased cell sensitivity to HuArgI (Co)-PEG5000 indicating that, following arginine deprivation, autophagy plays a protective role in GBM cells. Analysis of the type of cell death revealed a lack of AnnexinV staining and caspase activation in HuArgI (Co)-PEG5000-treated cells, indicating that arginine deprivation induces caspase-independent, non-apoptotic cell death in GBM. We have shown that GBM cells are auxotrophic for arginine and can be selectively targeted using HuArgI (Co)-PEG5000-induced arginine depletion, thus demonstrating that L-Arginine deprivation is a potent and selective potential treatment for GBM.