In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b+ macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-β, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80+CD11c+CD206+ macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80+CD11c+ (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.