Lung cancer is the leading cause of cancer-related mortality in humans. Exploration of the mechanisms underlying the self-renewal and stemness maintenance of cancer stem-like cells (CSLCs) will open new avenues in lung cancer diagnosis and therapy. Here, we isolated and identified a subpopulation of lung cancer stem-like cells (LCSLCs) from non-small cell lung carcinoma (NSCLC) A549 cells with features including self-renewal capacity in vitro, elevated tumorigenic activity in vivo, and high expression of stemness markers CD44, CD133, aldehyde dehydrogenase 1 (ALDH1) and Sox2, using a serum-free suspension sphere-forming culture method. We then found a higher expression level of gonadotropin-releasing hormone (GnRH) in the LCSLCs using a microarray assay, suggesting that GnRH may play a role in the self-renewal capacity and stemness maintenance in lung cancer cells. In addition, the suppression of GnRH capacity negatively regulated self-renewal and stemness maintenance in the LCSLCs. Overexpression of GnRH promoted stemness properties of A549-derived LCSLCs, indicating that GnRH expression is essential for the self-renewal and stemness maintenance in LCSLCs. Moreover, further investigations demonstrated that the promotion of GnRH functions of self-renewal and stemness maintenance in LCSLCs was associated with the JNK signaling pathway. Therefore, our results showed that GnRH participates in the self-renewal capacity and stemness maintenance of LCSLCs by upregulating the JNK signaling pathway, and GnRH may be useful as an alternative LCSLC therapy.