Background <p>Bladder cancer (BCa) is the most frequently seen malignancy of the urinary tract. However, its molecular mechanisms and therapeutic targets are not well established.</p> Objective <p>This study aims to investigate the mechanism by which tissue specific transplantation antigen P35B (TSTA3) mediates the suppression of epithelial-mesenchymal transition (EMT) in BCa through targeted regulation of lysosome-associated membrane protein 2 (LAMP2) expression via the mitogen-activated protein kinase (MAPK) signaling pathway.</p> Methods <p>Public datasets were analyzed to predict TSTA3 expression and prognosis in BCa. TSTA3 and LAMP2 expression levels were examined in 30 paired BCa and adjacent normal tissues, followed by Pearson correlation analysis of their mRNA levels. TSTA3 expression was quantified in T24, BIU-87 and simian virus 40-immortalized human urothelial cell line-1 (SV-HUC-1). T24 and BIU-87 cells were subjected to TSTA3 knockdown or overexpression. Cell proliferation, migration/invasion (Transwell), apoptosis (flow cytometry), EMT markers (immunofluorescence), and LAMP2/MAPK pathway proteins were evaluated.</p> Results <p>TSTA3 upregulation was demonstrated in public databases, BCa patient tissues, and cell strains. TSTA3 knockdown in T24 cells substantially suppressed proliferation, colony formation, invasion, migration, and apoptosis while increasing E-cadherin and decreasing Vimentin expression, whereas TSTA3 overexpression in BIU-87 cells promoted malignant phenotypes. TSTA3 and LAMP2 mRNA levels showed a strongly negative correlation in BCa patients. LAMP2 knockdown reversed the tumor-suppressive effects of TSTA3 silencing. Inhibition of the MAPK pathway rescued the functional deterioration of T24 cells caused by TSTA3 overexpression.</p> Conclusions <p>TSTA3 promotes BCa proliferation, migration, invasion, and EMT by regulating LAMP2 to activate the MAPK pathway.</p> Graphical abstract <p></p>