Combination therapy, using agents that target the microenvironment as well as the cancer cells, is common in the treatment of advanced breast cancer. Here, we show that a 6-week course of weekly sequential administration of the cytotoxic drug doxorubicin (2 mg/kg), followed 24 hr later by the antiresorptive agent zoledronic acid (100 μg/kg), causes substantial inhibition of subcutaneous MDA-MB-436 breast tumor growth in immunocompromised mice, leading to significantly increased survival. Tumor growth did not resume following withdrawal of treatment after 6 weeks, with 60% of the animals in this group surviving for more than 160 days. In comparison, animals receiving single-agent therapy all died within 50 days. Molecular analysis of the tumors showed no effect on cell cycle or apoptosis following administration of 100 μg/kg zoledronic acid or 2 mg/kg doxorubicin alone. When doxorubicin was administered 24 hr before zoledronic acid, tumors displayed decreased expression of CYCLINS E1, B, D1 and D3 as well as CDK2, CDC2, CDK4 and CDK7, indicative of cell-cycle inhibition. Tumors from animals receiving sequential treatment also showed induction of both intrinsic- and extrinsic-apoptotic pathways, with increased expression of BAX, decreased expression of BCL-2 and activation of CASPASE 3, 8 and 9. Accumulation of the unprenylated form of RAP1a, a surrogate marker for uptake of zoledronic acid, was only detected in tumors from animals treated with doxorubicin 24 hr before zoledronic acid. Our data are the first to show a sustained antitumor effect in vivo following a limited course of sequential administration of doxorubicin followed by zoledronic acid.