microRNAs (miRNAs) have gained more attention due to the biological functions in many cancers, including non-small cell lung cancer (NSCLC). However, the roles and the mechanism of miR-140-3p in NSCLC progression remain poorly understood. In this study, the expression levels of miR-140-3p and Janus kinase 1 (JAK1) were measured in NSCLC tissues and cells by quantitative real-time PCR. Cell viability, apoptosis, migration and invasion were detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-trtrazolium bromide, flow cytometry, Western blot or trans-well assay, respectively. Murine xenograft model was conducted to analyze the anti-tumor effect of miR-140-3p in vivo. Interaction between miR-140-3p and JAK1 was probed by luciferase reporter activity and Western blot. We found that miR-140-3p expression was down-regulated and JAK1 expression was increased in NSCLC tissues and cells compared with those in corresponding controls. Moreover, overexpression of miR-140-3p inhibited cell viability, migration and invasion while promoted cell apoptosis in NSCLC cells and suppressed NSCLC xenograft tumor growth in vivo. Besides, JAK1 was proved as a target of miR-140-3p and its restoration reversed miR-140-3p-mediated regulatory effect on progression of NSCLC. We concluded that miR-140-3p inhibited NSCLC progression by targeting JAK1, providing a novel avenue for treatment of NSCLC.