Despite advancements in therapeutic options, the overall prognosis for non-small-cell lung cancer (NSCLC) remains poor. Further exploration of the etiology and targets for novel treatments is crucial for managing NSCLC. In this study, we revealed the significant potential of EPB41 for inhibiting NSCLC proliferation, invasion and metastasis in vitro and in vivo. Consistent with its tumor suppressor role in NSCLC, the expression of EPB41 in NSCLC specimens evidently decreased compared to that in normal tissues, and low EPB41 expression was associated with poor prognoses for NSCLC patients. We further demonstrated the importance of EPB41 protein as a novel inhibitor of the Wnt signaling, which regulates beta-Catenin stability, and elucidated the crucial role of the EPB41/ALDOC/GSK3 beta/beta-Catenin axis in NSCLC. Suppression of EPB41 expression in cancer cells elevated the levels of free ALDOC protein released from the EPB41-ALDOC complex, leading to disassembly of the beta-catenin destruction complex, reduced proteolytic degradation of beta-catenin, elevated cytoplasmic accumulation and nuclear translocation of beta-catenin, thereby activating the expression of multiple oncogenes and, thus, NSCLC pathogenesis. Our study highlights the potential of EPB41 as a future therapeutic target for lung cancer.