In this study, we attempt to target Arginine auxotrophy in colorectal cancer cell lines 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 seven colorectal cancer cell lines. HuArgI (Co)-PEG5000 was cytotoxic to the majority of colorectal cancer cell lines tested (four out of seven). Addition of L-citrulline led to the rescue of all the sensitive colorectal cancer cell lines at concentrations of 11.4 mM, 1.14 mM but not at 0.114 mM. The ability of L-citrulline to rescue cells was dependent on the expression of argininosuccinate synthetase-1 (ASS-1) where single cell intracellular staining and flow cytometry indicated that all the colon cancer cell lines tested do express (ASS-1) indicating that these cell lines are partially auxotrophic for arginine. Analysis of the type of cell death revealed an increase in fraction of cells positive for both AnnexinV and PI, in HuArgI (Co)-PEG5000-treated cells, indicating that arginine deprivation induces mostly a non-apoptotic, necrotic mode of cell death in colorectal cancer cells. Inhibition of autophagy decreased cell sensitivity to HuArgI (Co)-PEG5000 indicating that, following arginine deprivation, autophagy is activated and may constitute the mechanism of arginine depletion-induced cell death in colorectal cancer cells. All the colon cancer cell lines tested showed signs of cell cycle arrest following HuArgI (Co)-PEG5000-induced arginine depletion, independently of their sensitivity to its cytotoxic effects. This confirms the presence of dual cytotoxic and a cytostatic roles of HuArgI(Co)-PEG5000-induced arginine depletion in colorectal cancer cell lines. We have shown that colorectal cancer 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 colorectal cancer.