Physical activity or exercise are known to promote bone formation and decrease bone resorption to maintain skeletal and bone health both in animal models and in humans with osteoporosis. Previous studies have indicated that long non-coding RNAs (lncRNAs) are able to regulate bone metabolism. Therefore, the present study aimed to evaluate whether lncRNAs responded to exercise by regulating the balance of bone metabolism in order to prevent osteoporosis. To meet this end, ovariectomized mice were used in the present study to establish an osteoporosis model. The exercise treatment groups were subjected to 9 weeks of treadmill running exercise in 4 weeks of the operation was performed Femurs were collected to measure bone mineral density, bone mass, bone formation and resorption. The expression levels of lncRNAs were subsequently measured using microarray and gene function analyses. The pairwise comparison results [ovariectomy (OVX) vs. OVX + exercise (EX); OVX vs. SHAM; SHAM vs. SHAM + EX; OVX + EX vs. SHAM + EX] of the gene microarray analysis revealed that the expression of 2,424 lncRNAs (1718 upregulated and 706 downregulated) were significantly altered in the mouse femurs following treadmill running. Gene Ontology (GO) analysis, incorporating the GO annotations 'biological processes', 'molecular function' and 'cellular components', of osteoporosis revealed that the VEGF, mTOR and NF-κB signaling pathways were potential targets of the lncRNAs. Moreover, it was possible to predict the target microRNAs (miRNAs) of six lncRNAs (LOC105246953, LOC102637959, NONMMUT014677, NONMMUT027251, ri|D130079K21|PX00187K16|1491 and NONMMUT006626), which suggested that the underlying mechanism by which lncRNAs respond to exercise involved bone regulation via lncRNA-miRNA sponge adsorption. Overall, these results suggested that the treadmill running exercise did regulate lncRNA expression in the bone, and that this was involved in the prevention of osteoporosis.