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Doxorubicin (DOX) is an anthracycline widely used in cancer therapy and in particular in breast cancer treatment. The treatment with DOX appears successful, but it is limited by a severe cardiotoxicity. This work evaluated the in vitro and in vivo anticancer effect of a new formulation of β-cyclodextrin nanosponges containing DOX (BNS-DOX). The BNS-DOX effectiveness was evaluated in human and mouse breast cancer cell lines in vitro in terms of effect on cell growth, cell cycle distribution, and apoptosis induction; and in vivo in BALB-neuT mice developing spontaneous breast cancer in terms of biodistribution, cancer growth inhibition, and heart toxicity. BNS-DOX significantly inhibited cancer cell proliferation, through the induction of apoptosis, with higher efficiency than free DOX. The breast cancer growth in BALB-neuT mice was inhibited by 60% by a BNS-DOX dose five times lower than the DOX therapeutic dose, with substantial reduction of tumor neoangiogenesis and lymphangiogenesis. Biodistribution after BNS-DOX treatment revealed a high accumulation of DOX in the tumor site and a low accumulation in the hearts of mice. Results indicated that use of BNS may be an efficient strategy to deliver DOX in the treatment of breast cancer, since it improves the anti-cancer effectiveness and reduces cardiotoxicity.