Arginine depletion interferes with pyrimidine metabolism and DNA damage repair pathways. Preclinical data demonstrated that depletion of arginine by PEGylated arginine deiminase (ADI-PEG 20) enhanced liposomal doxorubicin (PLD) cytotoxicity in cancer cells with argininosuccinate synthase 1 (ASS1) deficiency. The objective of this study was to assess safety and tolerability of ADI-PEG 20 and PLD in patients with metastatic solid tumors.Patients with advanced ASS1-deficient solid tumors were enrolled in this phase 1 trial of ADI-PEG 20 and PLD following a 3 + 3 design. Eligible patients were given intravenous PLD biweekly and intramuscular (IM) ADI-PEG 20 weekly. Toxicity and efficacy were evaluated according to the Common Terminology Criteria for Adverse Events (version 4.0) and Response Evaluation Criteria in Solid Tumors (version 1.1), respectively.Of 15 enrolled patients, 9 had metastatic HER2-negative breast carcinoma. We observed no dose-limiting toxicities or treatment-related deaths. One patient safely received 880 mg/m2 PLD in this study and 240 mg/m2 doxorubicin previously. Treatment led to stable disease in 9 patients and was associated with a median progression-free survival time of 3.95 months in 15 patients. Throughout the duration of treatment, decreased arginine and increased citrulline levels in peripheral blood remained significant in a majority of patients. We detected no induction of anti-ADI-PEG 20 antibodies by week 8 in one third of patients.Concurrent IM injection of ADI-PEG 20 at 36 mg/m2 weekly and intravenous infusion of PLD at 20 mg/m2 biweekly had an acceptable safety profile in patients with advanced ASS1-deficient solid tumors. Further evaluation of this combination is under discussion.© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.