Cancer immunotherapies, including immune checkpoint blockade, have the potential to significantly impact treatments for diverse tumor types. At present, response failures and immune-related adverse events remain significant issues, which could be addressed using optimized combination therapies. Through a cell-based chemical screen of ∼200,000 compounds, we identified that HSP90 inhibitors robustly decrease PD-L1 surface expression, through a mechanism that appears to involve the regulation of master transcriptional regulators (i.e., STAT-3 and c-Myc). Interestingly, HSP90 inhibitors were found to also modulate the surface expression of additional checkpoint proteins (i.e., PD-L2). In the MC-38 syngeneic mouse tumor model, HSP90 inhibition was found to dramatically reduce PD-L1 surface expression on isolated live tumor cells and, consistent with recent findings, was found to increase the number of activated CD8+ T cells within the tumor microenvironment. These findings provide further rationale to explore HSP90 inhibitors as part of combination immunotherapies for the treatment of cancer.