Objective To explore the possibility of microRNA miR-31-3p as a biomarker for bone metastasis of non-small-cell lung cancer (NSCLC) and its molecular mechanism to the invasion and metastasis of NSCLC cells. Methods Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of miR-31-3p and forkhead box 1 (FOXO1) in NSCLC tissues, serum, and cells to analyze the correlation between the expression levels of miR-31-3p and the clinicopathology of NSCLC. After interference with or overexpressing miR-31-3p, NSCLC cell proliferation, apoptosis, invasion ability, and migration ability were detected by MTT, flow cytometry, Transwell, and scratch experiment, respectively. The interaction between miR-31-3p and FOXO1 was further verified by the dual-luciferase reporter experiment. Western blot was performed to detect the protein expression of FOXO1 in tissues and FOXO1, RhoA, p-RhoA, ROCK-2, and p-ROCK-2 in cells. Results In tissues, serum, and NSCLC cell line A549 of the NSCLC patients, the expression of FOXO1 was notably lower, and the miR-31-3p expression was significantly higher. Overexpression of miR-31-3p could distinctly improve the proliferation, invasion, and migration of A549 cells, meanwhile inhibit cell apoptosis, and activate the RhoA/ROCK-2 signaling pathway, while interfering with the expression of miR-31-3p has the opposite function. Besides, bioinformatics analysis and luciferase reporter assay confirmed that FOXO1 was a target gene of miR-31-3p. Overexpressing FOXO1 could inhibit the proliferation and metastasis of A549 cells, but overexpressing miR-31-3p reverses the results. Conclusion This study confirmed that miR-31-3p promotes the proliferation, invasion, and migration of NSCLC cells and inhibits apoptosis through targeted regulating FOXO1 and be a potential therapeutic targets for the treatment of NSCLC.