In vitro studies have shown that hydrogen peroxide (H2O2) produced by high-concentration ascorbate and cell culture medium iron efficiently kills cancer cells. This provided the rationale for clinical trials of high-dose intravenous ascorbate-based treatment for cancer. A drawback in all the in vitro studies was their failure to take into account the in vivo concentration of iron to supplement cell culture media which are characterized by low iron content. Here we showed, using two prostate cancer cell lines (LNCaP and PC-3) and primary astrocytes, that the anticancer/cytotoxic effects of ascorbate are completely abolished by iron at physiological concentrations in cell culture medium and human plasma. A detailed examination of mechanisms showed that iron at physiological concentrations promotes both production and decomposition of H2O2. The latter is mediated by Fenton reaction and prevents H2O2 accumulation. The hydroxyl radical, which is produced in the Fenton reaction, is buffered by extracellular proteins, and could not affect intracellular targets like H2O2. These findings show that anticancer effects of ascorbate have been significantly overestimated in previous in vitro studies, and that common cell culture media might be unsuitable for redox research.