CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.