Adenylate kinase 2 (AK2), an isoenzyme of the AK family, may have momentous extra-mitochondrial functions, especially in tumourigenesis in addition to the well-known control of energy metabolism. In this study, we provided the first evidence that AK2 is overexpressed in lung adenocarcinoma. The positive expression of AK2 is associated with tumor progression, and poor survival in patients with pulmonary adenocarcinoma. Knockdown of AK2 could suppress proliferation, migration, and invasion as well as induce apoptosis and autophagy in human lung adenocarcinoma cells. Remarkably, silencing AK2 exerted the greater tumor suppression roles when combined with hydroxychloroquine, an effective autophagy inhibitor, in vitro and in xenografts mouse models. Our data have probably provided preclinical proof that systematic inhibition of AK2 and autophagy could be therapeutically effective on lung cancer.