Adenocarcinoma of Non-Small Cell Lung Cancer (NSCLC) is a severe disease. Patients carrying EGFR mutations may benefit from EGFR targeted therapies (e.g.: gefitinib). Recently, it has been shown that sialidase NEU3 directly interacts and regulates EGFR. In this work, we investigate the effect of sialidase NEU3 overexpression on EGFR pathways activation and EGFR targeted therapies sensitivity, in a series of lung cancer cell lines. NEU3 overexpression, forced after transfection, does not affect NSCLC cell viability. We demonstrate that NEU3 overexpression stimulates the ERK pathway but this activation is completely abolished by gefitinib treatment. The Akt pathway is also hyper-activated upon NEU3 overexpression, but gefitinib is able only to decrease, and not to abolish, such activation. These findings indicate that NEU3 can act directly on the ERK pathway through EGFR and both directly and indirectly with respect to EGFR on the Akt pathway. Furthermore, we provide evidence that a healthy mucosa cell line (with EGFR wild-type gene sequence) is slightly sensitive to gefitinib, especially in the presence of NEU3 overexpression, thus hypothesizing that NEU3 overexpressing patients may benefit from EGFR targeted therapies also in absence of EGFR point mutations. Overall, the expression of NEU3 may be a novel diagnostic marker in NSCLC because, by its ability to stimulate EGFR downstream pathways with direct and indirect mechanisms, it may help in the identification of patients who can profit from EGFR targeted therapies in absence of EGFR activating mutations or from new combinations of EGFR and Akt inhibitors.