Background <p>Mesothelin (MSLN), a cell surface glycoprotein, is commonly expressed in several cancers, including 40% of triple negative breast cancer (TNBC) cases. Although its cellular or physiologic functions remain unclear, MSLN has been leveraged as a target for molecular therapies. This study investigates MSLN’s potential role in TNBC, the breast cancer (BC) subtype with poorest outcomes.</p> Patients and Methods <p>The breast cancer (BC) cohorts from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, <i>n</i> = 313), The Cancer Genome Atlas (TCGA, <i>n</i> = 160), and SCAN-B (<i>n</i> = 155) were used to obtain biological variables and gene expression data.</p> Results <p>High MSLN expression was primarily observed in epithelial cells, and its expression was associated with reduced stromal adipocytes in the tumor microenvironment (TME), supporting its role as a TNBC surface marker. MSLN expression was also associated with the TNBC subtype and advanced N stages in BC. However, MSLN expression did not correlate with long-term survival outcomes, including overall survival (OS), disease-specific survival (DSS), or disease-free survival (DFS); nevertheless, it was still associated with favorable response to neoadjuvant chemotherapy (NAC). Indeed, MSLN-high tumors exhibited a proinflammatory microenvironment, higher cancer-testis antigen (CTA) scores, and increased immune cell activity, notably immature dendritic cells (iDCs) and M1 macrophages. Biologically, MSLN expression was linked to increased cellular proliferation, with gene set enrichment analysis (GSEA) showing enrichment in pathways related to rapid proliferation, such as G2M checkpoint and E2F targets.</p> Conclusions <p>MSLN-high TNBC is characterized by increased tumor grade, enhanced cell proliferation, and a proinflammatory microenvironment, showing better response to neoadjuvant chemotherapy without significant impact on long-term survival outcomes.</p>