Proprotein convertases (PCs) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether protein convertase enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. While altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacological inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) repressed PD-1 and exhaustion of CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable (MSI) and stable (MSS) colon cancer cells. In vivo, inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared to immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NFκB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy.