<p>Dysregulation of Eph receptor-ephrin signaling contributes to tumorigenesis, yet the specific role of Ephrin B3 in cutaneous squamous cell carcinoma (cSCC) remains poorly understood. This study identifies Ephrin B3 as a critical oncogenic driver in cSCC, demonstrating its significant overexpression in cSCC tissues and association with poor prognosis. To elucidate its tumor-promoting mechanisms, we generated Ephrin B3 knockout (<i>Efnb3</i>^−/−) and wild-type (<i>Efnb3</i>^WT) mouse models of DMBA/TPA-induced skin carcinogenesis. Strikingly, Ephrin B3 deletion significantly inhibited cSCC carcinogenesis, while its knockdown in A431 human cSCC cells inhibited proliferation, migration, and invasion, underscoring its pivotal role in tumor aggressiveness. Integrative label-free proteomic analysis revealed cytokeratin 19 (CK19) as the most differentially expressed protein in <i>Efnb3</i>^WT versus <i>Efnb3</i>^−/− mice, establishing a novel molecular connection between Ephrin B3 and CK19. Further mechanistic studies demonstrated that Ephrin B3 positively regulated the NOTCH1 signaling pathway through CK19. Additionally, Reactome pathway analysis implicated Ephrin B3 in inflammation-mediated carcinogenesis through the MAPKs pathway. Consistent with this, <i>Efnb3</i> deficiency mitigated inflammatory responses in an acute skin inflammation model, suggesting its role in shaping a pro-tumorigenic microenvironment. Collectively, these findings not only establish Ephrin B3 as a potential prognostic biomarker for cSCC but also reveal its&#xa0;dual mechanistic role in fostering tumorigenesis via the CK19-NOTCH1 axis and amplifying inflammation through MAPKs signaling. This study provides groundbreaking insights into multifaceted contributions of Ephrin B3 in cSCC pathogenesis and opens new avenues for targeted therapeutic intervention.</p>