Tumor hypoxia remains one of the greatest challenges in the treatment of solid tumors, as cancer cells in these regions are resistant to killing by radiation therapy and most anticancer drugs. Tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide or SR 4233) is a cytotoxic drug with selective toxicity towards hypoxic mammalian cells. Under both aerobic and hypoxic conditions, tirapazamine is reduced by an intracellular reductase to form a highly reactive radical, which can cause DNA single- and double-strand breaks. In addition, tirapazamine under hypoxic conditions reduces the activity of topoisomerase II and stabilizes DNA topoisomerase II cleavable complexes, and these complexes remain bound to DNA. Despite the very promising results obtained in various preclinical studies and early-Phase clinical trials, several Phase III trials have failed to demonstrate any survival benefit of adding tirapazamine to chemotherapy or radiation therapy of cancers.