In our study, the impact of miR-16-1-3p on cell proliferation and invasion in NSCLC was explored. miR-16-1-3p mimics were transfected to A549 cells for miR-16-1-3p overexpression. qRT- PCR and Western blot were applied to explore the relative expression of mRNA and protein in A549 cells. Furthermore, the cell proliferation capability was determined by MTT assay. Additionally, cell migration and invasion were measured using a scratch assay and transwell assay, respectively. Moreover, TargetScan and luciferase reporter assay was utilized to investigate the target of miR-16-1-3p. The results indicated that miR-16-1-3p was downregulated in NSCLC cells and upregulation of miR-16-1-3p was able to inhibit the expression of TWIST1. In addition, the reduced cell proliferation, inhibited cell migration and invasion were observed in miR-16-1-3p mimic group compared to the negative control group. The luciferase reporter gene showed that TWIST1 was a target of miR-16-1-3p. Therefore, the present study demonstrated that miR-16-1-3p may suppress A549 cell proliferation, migration and invasion by targeting TWIST1. Thus, miR-16-1-3p might play important roles in NSCLC development, which provides a novel aspect for NSCLC investigation (Fig. 6, Ref. 26).