High-dose vitamin C (VC) exhibits anti-tumor effects, and the cytotoxicity of VC is correlated with oxidative stress. However, iron, as a redox metal, plays an important effect in redox cycling and free radical formation in cells. This study addresses the role of iron ion in the cytotoxicity of VC. We found that iron supplementation increases the anti-tumor effect of VC, which was influenced by the cellular iron uptake pathway–transferrin (TF)/transferrin receptor (TFR) system. The TFR expression of tumors can be assessed by ⁶⁸Ga-citrate PET imaging, and it would be helpful to screen out the tumor type which is more sensitive to VC combined with an iron supplementation treatment.