Zoledronic acid (ZOL), a nitrogen‑containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti‑leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti‑leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL‑60 and adriamycin‑resistant HL‑60 (HL‑60/A) cells using a WST‑8 assay. An Annexin V‑propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose‑ and time‑dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S‑phase cell cycle arrest, downregulation of B‑cell lymphoma 2 (Bcl‑2) and upregulation of Bcl‑2 associated X protein and cleaved poly (ADP‑ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti‑leukemic activity appeared notably enhanced in HL‑60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.