The vast research and clinical result have verified the success of cancer immunotherapy. However, there is also facing the enormous challenges such as lack of precise pre-clinical models, optimal combined therapy regimen and acquired resistance to immunotherapy. Adenosine is a potent immune-modulating molecule and overexpression of CD73 on tumor leads to the high concentration of adenosine. Blockade of the key adenosine-generating enzyme CD73 can be a promising strategy for cancer immunotherapy. Here, we report the discovery of betulinic acid as a novel CD73 inhibitor lead compound by a hit-based substructure search strategy. Subsequent optimization led to the discovery of betulinic acid carbamate derivative ZM514 with 5.2-fold increased potency compared to lead compound. Simultaneously, study has showed that compound ZM514 was not a cytotoxic agent while betulinic acid showed modest antiproliferative activity. The present result provides a valuable inhibitor against the promising immuno-oncology target for further development.