Hypoxia, frequently found in the center of solid tumors, may lead to enhance the production of key factor in cell survival, invasion, angiogenesis and loss of apoptosis. The low oxygen tension in hypoxic tumors is also known to interfere with the efficacy of chemotherapy, but the underlying mechanisms are not very clear. Paclitaxel (PTX) is an active agent used in breast cancer chemotherapy, which disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. In the present study, we try to determine whether hypoxia can decrease the chemosensitivity of human breast carcinoma cells to PTX and elucidate the underlying mechanism. We found that hypoxia could decrease PTX-induced cell death and G₂/M arrest. Furthermore, our results showed that hypoxia inhibit PTX-induced soluble tubulin polymerized. In addition, we also found hypoxia could suppress PTX-induced cell cycle protein—cyclin B1 expression in MCF-7 cells. To further investigate whether the inhibitory effect of hypoxia on PTX-induced cell death is mediated by decreasing levels of cyclin B1, cyclin B1-transfected MCF-7 cells were used under hypoxic condition. The data showed that the hypoxia-based decreasing chemosensitivity of breast cancer cells to PTX was reversed by cyclin B1. We also found that overexpression of cyclin B1 could significantly increase the sensitivity of MCF-7 cells to PTX by stimulating soluble polymerized tubulin. Overall, hypoxia decreases cyclin B1, which could in turn reverse hypoxia-induced decreasing chemosensitivity to PTX in breast cancer cell line MCF-7.