The present study aimed to identify the function of miR-491-3p in regulating non-small cell lung cancer (NSCLC). Tumor tissues and adjacent normal tissues were collected from 43 patients with NSCLC. A549 and H1299 cells were transfected with microRNA (miR)-491-3p mimic, mimic negative control (NC), miR-491-3p inhibitor, inhibitor NC, pcDNA3.1-FGF5 vector and control vector. Cell counting kit-8 assay and Edu experiments were performed to assess cell viability and proliferation. Matrigel experiment, wound healing assay and flow cytometric analysis were performed to explore cell invasion, migration and apoptosis, respectively. A dual-luciferase reporter experiment was performed to identify the relationship between miR-491-3p and fibroblast growth factor 5 (FGF5). In vivo study was conducted by using nude mice. The miR-491-3p and FGF5 protein expression levels were investigated using reverse transcription-quantitative polymerase chain reaction and western blot analysis. In NSCLC tumor tissues, miR-491-3p was downregulated and FGF5 was upregulated (P<0.01). Low miR-491-3p expression and high FGF5 mRNA expression was associated with poor outcomes in patients, including advanced TNM stage and lymph node metastasis (P<0.05). upregulation of miR-491-3p suppressed viability, proliferation, invasion and migration of NSCLC cells; however, it promoted apoptosis (P<0.01). FGF5 was a target gene for miR-491-3p. miR-491-3p directly inhibited FGF5 expression. upregulation of FGF5 significantly reversed the inhibitory effects of miR-491-3p on malignant phenotypes of NSCLC cells (P<0.01). miR-491-3p overexpression suppressed the in vivo growth of NSCLC. Thus, it was identified that miR-491-3p functions as a tumor suppressor in NSCLC by directly targeting FGF5.