The conformation-dependent activity of azobenzene combretastatin A4 (Azo-CA4) provides a unique approach to reduce the side-effects of chemotherapy, due to the light-triggered conformation transition of its azobenzene moiety. Under hypoxic tumor microenvironment, however, the high expression of azoreductase can reduce azobenzene to aniline. It was postulated that the Azo-CA4 might be degraded under hypoxia, resulting in the decrease of its anti-tumor activity. The aim of this study was to verify such hypothesis in HeLa cells . The quantitative drug concentration analysis shows the ratiometric formation of degradation end-products, confirming the bioreduction of Azo-CA4. The tubulin staining study indicates that Azo-CA4 loses the potency of switching off microtubule dynamics under hypoxia. Furthermore, the cell cycle analysis shows that the ability of Azo-CA4 to induce mitotic arrest is lost at low oxygen content. Therefore, the cytotoxicity of Azo-CA4 is compromised under hypoxia. In contrast, combretastatin A4 as a positive control maintains the potency to inhibit tubulin polymerization and break down the nuclei irrespective of light irradiation and oxygen level. This work highlights the influence of hypoxic tumor microenvironment on the anti-tumor potency of Azo-CA4, which should be considered during the early stage of designing translational Azo-CA4 delivery systems.