L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H 2 O 2 , which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H 2 O 2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H 2 O 2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H 2 O 2 -scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo . The addition of AUR to L-ASC-treated cells triggers the accumulation of H 2 O 2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings. fx1 • Lack of peroxiredoxin 1 potentiates antileukemic activity of L-ascorbate in vitro and in vivo. • Auranofin and L-ascorbate synergistically kill malignant B cells. • Auranofin leads to intracellular accumulation of H 2 O 2 generated by L-ascorbate. • Auranofin and L-ascorbate trigger iron-dependent oxidative damage and cytotoxicity.