MicroRNAs are a class of noncoding RNAs that can be involved in the regulation of gene expression in cancers, including lung cancer. Our previous research has shown that miR-486-5p is one of the most downregulated microRNAs in tissue and serum samples of lung cancer as a good diagnostic biomarker. The objective of this study is to investigate the roles of miR-486-5p in the progression of lung cancer. In this study, miR-486-5p was further validated to be significantly downregulated in additional nonsmall cell lung cancer (NSCLC) tissue, serum, and cell samples by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression level of miR-486-5p was significantly associated with clinical phenotype of NSCLC. The PIK3R1 gene was confirmed to be a direct target of miR-486-5p by dual-luciferase reporter assay, and the expression level of miR-486-5p was inversely correlated with that of PIK3R1 in tumor tissues ( = -0.774, < 0.01). Overexpressed miR-486-5p effectively inhibited cell proliferation and invasion and successfully induced apoptosis . PIK3R1 was involved in the suppression of miR-486-5p on cell growth. It can be concluded that miR-486-5p may act as a tumor suppressor contributing to the progression of NSCLC, and miR-486-5p would be a diagnostic and prognostic biomarker and a potential therapeutic target for lung cancer.