Glioma is extremely difficult to be completely excised by surgery due to its invasive nature. Thus, chemotherapy still is the mainstay in the treatment of glioma after surgery. However, the natural blood-brain barrier (BBB) greatly restricts the penetration of chemotherapeutic agents into the central nervous system. As a front-line anti-glioma agent in clinical, carmustine (BCNU) exerts antitumor effect by inducing DNA damage at the O ⁶ position of guanine. However, the therapeutic effect of BCNU was largely decreased because of the drug resistance mediated by O ⁶ -alkylguanine-DNA alkyltransferase (AGT) and insufficient local drug concentrations. To overcome these obstacles, we synthesized a BCNU-loaded hypoxia-responsive nano-micelle with BBB penetrating capacity and AGT inhibitory activity, named as T80-HA-AZO-BG/BCNU NPs. In this nano-system, Tween 80 (T80) serves as a functional coating on the surface of the micelle, promoting transportation across the BBB. Hyaluronic acid (HA) with active tumor-targeting capability was linked with the hydrophobic O ⁶ -benzylguanine (BG) analog via a hypoxia-sensitive azo bond. Under hypoxic tumor microenvironment, the azo bond selectively breaks to release O ⁶ -BG as AGT inhibitor and BCNU as DNA alkylating agent. The synthesized T80-HA-AZO-BG/BCNU NPs showed good stability, favorable biocompatibility and hypoxia-responsive drug-releasing ability. T80 modification improved the transportation of the micelle across an in vitro BBB model. Moreover, T80-HA-AZO-BG/BCNU NPs exhibited significantly enhanced cytotoxicity against glioma cell lines with high AGT expression compared with traditional combined medication of BCNU plus O ⁶ -BG. We expect that the tumor-targeting nano-micelle designed for chloroethylnitrosourea will provide new tools for the development of effective glioma therapy.