The expression and functions of microRNA (miR)-320 have been previously investigated in various types of cancer. However, to the best of our knowledge, no previous studies have investigated miR-320 in human lung cancer. The current study determined the expression, biological functions and molecular mechanisms of miR‑320 in human lung cancer. The expression level of miR‑320 in human non‑small cell lung cancer (NSCLC) and normal adjacent tissue samples (NATs), NSCLC cell lines and non‑tumorigenic bronchial epithelial cells was measured by reverse transcription‑quantitative polymerase chain reaction. Following transfection with miR‑320 mimics, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide, cell migration and cell invasion assays, western blot analysis and luciferase assay were performed in human NSCLC cell lines. The results demonstrated that miR‑320 was significantly downregulated in NSCLC tissue samples and cell lines compared with NATs and a control cell line, respectively. Statistical analysis demonstrated that expression of miR‑320 was significantly associated with the TNM classification and metastasis. It was also observed that miR‑320 inhibited cell growth, migration and invasion in NSCLC cells. Additionally, the present study provided evidence that miR‑320 may directly target fatty acid synthase. These results suggest that miR‑320 may serve as a therapeutic biomarker of NSCLC in the future.