Objective To investigate the effect of AS-miR-16-5p on the cell viability and migration of non-small cell lung cancer (NSCLC) A549 cells and its potential mechanism. Methods NSCLC A549 cells were divided into black group, Mir NC group and AS-Mir-16-5p group. Mir NC group and AS-Mir-16-5p group were transfected with negative control miRNA (MIR NC) and AS-Mir-16-5p respectively. Black group did not intervene. The expression of Mir-16-5p in A549 cells was detected by real-time quantitative polymerase chain reaction (RT qPCR). MTT assay was used to detect the activity of A549 cells. The migration ability of A549 cells was detected by scratch test and Transwell test. The expression of key proteins of PTEN / PI3K / Akt signaling pathway in A549 cells was detected by Western blotting. Results Compared with the miR-NC group and the Black group, the expression level of miR-16-5p in the AS-miR-16-5p group was downregulated (P＜0.05). AS-miR-16-5p significantly inhibited the viabilit of A549 cells (P＜0.05), significantly reduce the migration rate (P＜0.05) of A549 cells. AS-miR-16-5p significantly increased the expression level of PTEN protein in A549 cells (P＜0.05), and significantly down-regulated the expression of PI3K and AKT protein (P＜0.05). Conclusion AS-miR-16-5p can inhibit the cell viability and migration of NSCLC A549 cells. In addition, AS-miR-16-5p negatively regulates the PTEN/PI3K/AKT signaling pathway and may become a new target for NSCLC therapy.