Abstract: Over recent years, immunotherapy has developed rapidly, which has changed the way of cancer treatment. However, most patients cannot benefit from immunotherapy, because of insufficient reprogramming of the immunosuppressive tumor microenvironment (TME) which thus limits reinvigoration of antitumor immunity. In TME, adenosine, a metabolite of ATP, is an effective immunoregulatory factor. Extracellular 5'-nucleotidase (CD73) is the rate-limiting molecule in the process of adenosine production. Overexpression of CD73 on tumor cells and immune cells leads to a higher concentration of adenosine in the TME. The high concentration of adenosine suppresses the anti-tumor immune response, promoting tumor cells proliferate, metastasis and angiogenesis. Therefore, anti-CD73 therapy is expected to become a promising strategy for cancer immunotherapy. Several anti-CD73 mAbs (MEDI9447, BMS986179, SRF373/NZV930, CPI-006/CPX-006, IPH5301, TJ004309) and small molecule CD73 inhibitors ((LY3475070, AB680, CB-708) were investigated in early phase clinical trials. But so far, there is no CD73 targeted product for the treatment of cancer on the market. In this study, we performed the screening of our compound library containing 876 listed drugs to identify candidate inhibitors targeting CD73. Preliminary experimental results showed that Cisatracurium besylate (51w89) could inhibit the enzyme activity of recombinant CD73 with an IC₅₀ value of 13.30 μmol/L. To verify the interactions between 51w89 and CD73 and evaluate their binding affinities, we performed surface plasmon resonance (SPR) experiments using a Biacore T200 (GE Healthcare). The binding affinity (K_D) of 51w89 binding to CD73 was 20.45 μmol/L. Encouraged by these results at the molecular level, then we evaluated the inhibitory effect of 51w89 against CD73 in MDA-MB-231 cells. The results show that the IC₅₀ of 51w89 against CD73 in MDA-MB-231 cells was (17.70±0.98) μmol/L, closing to the IC₅₀ value at the molecular level. Subsequently, we proved the role of CD73 in the migration of MDA-MB-231 cells through scratch tests, transwell tests and siRNA tests. The results demonstrated that 51w89 could inhibit the migration of MDA-MB-231 cells. Moreover, we found that 51w89 could limit the inhibitory effect of AMP on CD8⁺T cells. Taken together, we speculated that 51w89 could be used as a potent small-molecule inhibitor of CD73 for subsequent antitumor research.