Anlotinib treatment of non-small cell lung cancer (NSCLC) is hindered by drug insensitivity. Downregulation of long non-coding RNA (lncRNA) NEAT1 can suppress the proliferation and invasion by NSCLC cells. This study explored the role of the combination of anlotinib with NEAT1 knockdown on NSCLC progression. A549 and NCI-H1975 cells were used to evaluate the effect of anlotinib with NEAT 1 knockdown on NSCLC cells in vitro. The proliferation, invasion, migration, and apoptosis of NSCLC cells were evaluated with CCK-8 assays, EdU staining, Transwell assays, and flow cytometry. The antitumor effect of anlotinib with NEAT 1 knockdown was further explored in a mouse xenograft model. NEAT 1 knockdown enhanced the inhibitory effect of anlotinib on NSCLC cell proliferation, migration, and invasion. NEAT 1 knockdown also increased the pro-apoptotic and cytotoxic effects of anlotinib through downregulation of the Wnt/β-catenin signaling pathway. The inhibitory effect of anlotinib on tumor growth was boosted in the presence of NEAT 1 knockdown in vivo. NEAT 1 knockdown promoted NSCLC cell sensitivity to anlotinib in vitro and in vivo. Thus, combined treatment of anlotinib with NEAT 1 knockdown may provide a new combined therapeutic approach for NSCLC patients.