Development of specific serum biomarkers is essential to improve diagnosis and prognosis of non-small cell lung cancer (NSCLC). Here, we show that serum and tissue levels of miR-519d are significantly decreased in NSCLC patients. The low expression of miR-519d is associated with lymph node metastases, clinical stage, and a poor prognosis in NSCLC patients. In addition, ROC analysis demonstrated that the serum miR-519d levels can distinguish NSCLC patients from healthy controls. MiR-519d inhibits proliferation, migration, and invasion by lung cancer cells, indicating that it may function as a tumor suppressor in lung cancer. Furthermore, our data demonstrate that HER3 is a target gene of miR-519d in lung cancer cells, and show that by targeting HER3, miR-519d inhibits the PI3K/Akt pathway. These findings demonstrate that the miR-519d levels are decreased in serum and tumor tissues of NSCLC patients, and indicate that miR-519d regulates NSCLC progression by targeting HER3. MiR-519d could potentially serve as a novel serum biomarker for NSCLC.