<p>The limb development membrane protein 1-like gene (LMBR1L) encodes a transmembrane protein involved in cellular signaling and molecular transport. LMBR1L plays a critical role in immune system development by negatively regulating the Wnt/β-catenin pathway in lymphocytes. However, its role in the tumor immune microenvironment and its potential as a predictor of immunotherapy response remain poorly understood. We analyzed LMBR1L expression in normal and tumor tissues using data from public databases and clinical samples. Prognostic significance and immune associations were assessed using survival analysis, Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA), and immune infiltration profiling tools. Additionally, both in vitro and in vivo experiments were conducted to assess the effects of LMBR1L knockdown in colorectal cancer (COAD) cells. Elevated LMBR1L expression was observed in multiple cancers. Single-cell sequencing revealed its primary expression in CD8⁺T cells, and higher expression was correlated with poorer outcomes. Increased LMBR1L levels were associated with immune cell infiltration, including CD4⁺and CD8⁺T cells, and correlated with programmed death-ligand-1 (PD-L1) expression, suggesting its involvement in immune checkpoint regulation. LMBR1L knockdown suppressed the proliferation, migration, and invasion of COAD cells, a finding further corroborated by in vivo experiments. Our findings suggest that LMBR1L may be a prognostic biomarker and an immunotherapy predictor, emphasizing its potential as a therapeutic target in cancer treatment. This study provides a foundation for future work on targeted therapy and immunotherapy strategies in cancer.</p>