The limited efficacy of vaccines in hepatocellular carcinoma (HCC), due to the low frequency of tumor-infiltrating cytotoxic T lymphocytes (CTLs), indicates the importance of innate immune surveillance, which assists acquired immunity by directly recognizing and eliminating HCC. Innate Vγ9Vδ2 T cells have major histocompatibility complex-unrestricted antitumor activity and are activated by phosphoantigens, which are upregulated in cancer cells by the nitrogen-containing bisphosphonate, zoledronate (Zol). A better understanding of HCC susceptibility to Zol and downstream γδ T cell-mediated killing is essential to optimize γδ T cell-mediated immunotherapy. This study systematically examined the interactions between γδ T cells and Zol-treated HCC cell lines (HepG2, HLE, HLF, HuH-1, JHH5, JHH7, and Li-7) in vitro. All HCC cell lines expressed the DNAX accessory molecule-1 ligands, poliovirus receptor, and Nectin-2, and γδ T cell-mediated killing of these cells was significantly enhanced by Zol. Small interfering RNA-mediated knockdown of these ligands did not affect the susceptibility to γδ T cell lysis. This killing activity was partly inhibited by mevastatin, an inhibitor of the mevalonate pathway, and markedly reduced by a monoclonal antibody to γ- and δ-chain T cell receptor, indicating that this is crucial for Zol-induced HCC killing. In addition, Zol-treated HCC cell lines triggered γδ T cell proliferation and induced production of Th1 and Th2, but not Th17, cytokines. The Zol concentration that enhanced HCC cell susceptibility to γδ T cell killing was lower than that required to directly inhibit HCC proliferation. Thus, γδ T cells may be important effector cells in the presence of Zol, especially where there are insufficient number of cancer antigen-specific CTLs to eliminate HCC. Our in vitro data support the proposal that Zol-treatment, combined with adaptive γδ T cell immunotherapy, may provide a feasible and effective approach for treatment of HCC.