The molecular mechanisms underlying tumour heterogeneity in bladder cancer remain to be explored. Here, the authors perform single cell RNA sequencing and identify CD39 as a potential target for immunotherapy, which they validate in vivo. Single-cell sequencing technologies have noteworthily improved our understanding of the genetic map and molecular characteristics of bladder cancer (BC). Here we identify CD39 as a potential therapeutic target for BC via single-cell transcriptome analysis. In a subcutaneous tumor model and orthotopic bladder cancer model, inhibition of CD39 (CD39i) by sodium polyoxotungstate is able to limit the growth of BC and improve the overall survival of tumor-bearing mice. Via single cell RNA sequencing, we find that CD39i increase the intratumor NK cells, conventional type 1 dendritic cells (cDC1) and CD8 + T cells and decrease the Treg abundance. The antitumor effect and reprogramming of the tumor microenvironment are blockaded in both the NK cells depletion model and the cDC1-deficient Batf3(-/-) model. In addition, a significant synergistic effect is observed between CD39i and cisplatin, but the CD39i + anti-PD-L1 (or anti-PD1) strategy does not show any synergistic effects in the BC model. Our results confirm that CD39 is a potential target for the immune therapy of BC.