Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.