Immune checkpoint blockade (ICB) therapy, targeting programmed cell death ligand-1 (PD-L1)/programmed cell death protein 1 (PD-1) axis and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), has exhibited amazing clinical outcomes in various types of cancers. However, only a small portion of patients benefit from ICB therapy, indicating that the mechanism underlying immune checkpoint is still unclear. Here, it is reported that motor neuron and pancreas homeobox 1 (MNX1), a homeobox domain-containing transcription factor, contributes to the tumor immune escape. MNX1 increases PD-L1 expression in cancer cells by stabilizing PD-L1 mRNA rather than activating transcription. Mechanistically, MNX1 exists in the cytoplasm of cancer cells and interacts with Y-box binding protein 1 (YBX1), a multifunctional DNA/RNA-binding protein, to enhance the binding of YBX1 to PD-L1 mRNA. MNX1 ablation activates cytotoxic T cell-mediated anti-tumor immunity and sensitizes CTLA-4 blockade therapy. Moreover, MNX1 also facilitates tumor progression in an immune-independent manner in cancer cells. In addition, MNX1 is upregulated by its adjacent long non-coding RNA MNX1-AS1 via HECT and RLD domain containing E3 ubiquitin protein ligase 2 (HERC2). Together, these results reveal MNX1 as a novel immune checkpoint regulator with promising therapeutic potential.