Relapse and refractoriness remain the major obstacles in clinical treatment of acute myeloid leukemia (AML). Efficacy of current therapeutic strategies for relapsed/refractory (R/R) AML is generally unsatisfying. Vδ2 T cells have become an attractive candidate for immunotherapy of various types of tumors. However, the results were not exciting in some pilot studies utilizing Vδ2 cell-based protocols to treat R/R AML. Functional receptors on Vδ2 cells and immunogenic ligands on leukemia cells are both critical to the anti-AML effect of Vδ2 cells, which have not been characterized in the context of R/R AML. CD277 can bind to phosphoantigens and promote the activation of Vδ2 cells. Anti-CD277 (clone 20.1) monoclonal antibody (20.1 mAb) has been identified as an agonist of CD277. Whether 20.1 mAb sensitizes R/R AML cells awaits investigation. Herein, we showed that the expressions of activating receptors on Vδ2 cells and CD277 on leukemia cells were deficient in patients with R/R AML. While agonists for NKG2D and TRAIL ligands did not increase the immunogenicity of R/R AML cells, 20.1 mAb significantly enhanced the cytotoxicity of Vδ2 cells on the drug-resistant human AML cell line and different types of primary AML cells from R/R patients. The sensitizing effect of 20.1 mAb was dependent on inducing degranulation of Vδ2 cells. These findings suggest a decisive role of CD277 in mediating the recognition of R/R AML cells by Vδ2 T cells. CD277 agonist combining adoptive transfer of Vδ2 T cells may improve the efficacy in the treatment of R/R AML.