Introduction: The anti-cancer effect of high concentrations of ascorbic acid (AA) has been well established while its underlying mechanisms remain unclear. The association between iron and AA has attracted great attention but was still controversial due to the complicated roles of iron in tumors. Objectives: Our study aims to explore the anti-cancer mechanisms of AA and the interaction between AA and iron in cancer. Methods: The MTT and ATP assays were used to evaluate the cytotoxicity of AA. Reactive oxygen species (ROS) generation, calcium (Ca), and lipid peroxidation were monitored with flow cytometry. Mitochondrial dysfunction was assessed by mitochondrial membrane potential (MMP) detection with JC-1 or tetramethylrhodamine methyl ester (TMRM) staining. Mitochondrial swelling was monitored with MitoTracker Green probe. FeSO (Fe), FeCl (Fe), Ferric ammonium citrate (Fe), hemin chloride (Fe) were used as an iron donor to investigate the effects of iron on AA's anti-tumor activity. The in vivo effects of AA and iron were analyzed in xenograft zebrafish and allograft mouse models. Results: High concentrations of AA exhibited cytotoxicity in a panel of cancer cells. AA triggered ROS-dependent non-apoptotic cell death. AA-induced cell death was essentially mediated by the accumulated intracellular Ca, which was partly originated from endoplasmic reticulum (ER). Surprisingly, exogenous iron could significantly reverse AA-induced ROS generation, Ca overloaded, and cell death. Especially, the iron supplements significantly impaired the in vivo anti-tumor activity of AA. Conclusions: Our study elucidated the protective roles of iron in ROS/Ca mediated necrosis triggered by AA both in vitro and in vivo, which might shed novel insight into the anti-cancer mechanisms and provide clinical application strategies for AA in cancer treatment.