<p>Zoledronic acid (ZA), a bisphosphonate, is commonly used in breast cancer patients with bone metastases to treat hypercalcemia and osteolysis. Recent studies showed the anti-cancer effects of ZA in breast cancer. This study further explored the synergistic effects of sequential and nonsequential ZA and doxorubicin (DOX) administration on estrogen receptor (ER)–positive and –negative breast cancer cell lines. Anti-cancer and anti-invasion effects were evaluated using MTT and Matrigel invasion assays. The synergistic effects were analyzed using the Chou-Talalay method. The protein levels of invasive and angiogenic factors were assessed by western blot. ZA was found to inhibit the proliferation of ER-positive and -negative breast cancer cells in a concentration-dependent manner. When ZA and doxorubicin (DOX) were sequentially combined at nontoxic concentrations, synergistic effects were observed in sequential administrations with DOX followed by ZA only in ER-negative breast cancer cells. Conversely, the sequential and nonsequential treatments did not significantly differ in ER-positive breast cancer cells. Moreover, this sequential treatment significantly reduced cell invasion and MMP9, pNF-κB, and FGF2 protein levels in ER-negative cells. The results suggest that ZA potentially inhibits ER-negative cells by suppressing the canonical NF-κB pathway and its downstream proteins, MMP9 and FGF2. Furthermore, DOX pretreatment enhanced the ZA effect and increased cell sensitivity to ZA, leading to improved outcomes with lower concentrations and shorter drug exposure durations. When combined with DOX, ZA produced synergistic effects on cell proliferation and invasion when administered sequentially in ER-negative breast cancer cells.</p>