Abstract Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T-cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of anti-tumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 costimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful anti-tumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell anti-tumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma.