Background <p>MUC3A is upregulated in the serum of patients with extrahepatic cholangiocarcinoma (EHCC). Nevertheless, the specific role of MUC3A in EHCC is elusive. Therefore, this study aimed to analyze the role and mechanism of MUC3A in cell proliferation and epithelial–mesenchymal transition (EMT) in EHCC.</p> Materials and Methods <p>UCHL1 and MUC3A expression levels were measured in EHCC cells and tissues. After gain- and loss-of-function assays in EHCC cells, cell proliferation was assessed with CCK-8, EdU, and colony formation assays, while cell invasion, migration, and apoptosis were examined with transwell, scratch, and flow cytometry assays, respectively. Western blot was implemented to detect the levels of migration- and EMT-related markers, as well as proteins related to the downstream pathways of EGFR. A xenograft tumor model was established for in vitro validation. The relationship between UCHL1 and MUC3A was analyzed with Co-immunoprecipitation (Co-IP), Western blotting, ubiquitination analysis, and immunofluorescence.</p> Results <p>UCHL1 and MUC3A were highly expressed in EHCC. MUC3A knockdown inhibited the viability, EMT, migration, and invasion of ECHH cells while accelerating their apoptosis. MUC3A knockdown repressed tumorigenesis of EHCC in mice. Mechanistically, UCHL1 acted as a deubiquitinase to maintain MUC3A stability and activated the downstream pathways of EGFR, namely Ras/Raf/MEK/ERK and PI3K/AKT. UCHL1 overexpression nullified the repressive impacts of MUC3A knockdown on EHCC cell viability, invasion, and EMT, while EGFR activation abrogated the suppressive impacts of UCHL1 depletion on EHCC cell viability, invasion, and EMT.</p> Conclusions <p>UCHL1 activates downstream pathways of EGFR by stabilizing MUC3A, thereby fostering EHCC progression.</p>