Non-small cell lung cancer (NSCLC) is a type of malignant tumor which threatens human health and life. Recently, some researches on long non-coding RNAs (lncRNAs) in NSCLC has elucidated critical regulatory roles in cell proliferation, migration, and invasion, the relative clinical significance and mechanisms of action are still unclear. This study focuses on the important role of a novel lncRNA LINC00665 in the development of NSCLC. Long intergenic non-protein coding RNA 665 gene (LINC00665) was found through microarray analysis and was measured by real-time quantitative PCR (RT-qPCR). The interactions between LINC00665 and miR-138-5p as well as the interactions between miR-138-5p and E2F3 (E2F transcription factor 3) were explored by bioinformatics analysis and dual-luciferase assays. CCK-8, transwell and mouse xenograft assays were performed to investigate the effects of LINC00665 and miR-138-5p on NSCLC proliferation and invasion. As a result, LINC00665 expression was upregulated in NSCLC lung tissues and cells. Downregulated LINC00665 could arrest A549 and H1299 cell proliferation and invasion in vitro, and this finding was recapitulated in vivo. LINC00665 directly regulated the expression of miR-138-5p. Additionally, E2F3 was one of the targets of miR-138-5p; E2F3 without 3′UTR could reverse the inhibitory effects of downregulated LINC00665 on proliferation and invasion in A549 and H1299 cells. In conclusion, dysregulation of LINC00665 plays a vital role in NSCLC progression, indicating that its downregulation may confer decreased cell proliferation and invasion via the miR-138-5p/E2F3 signaling pathway.