Osteoarthritis is a common disease character with progressive destruction of cartilage. MicroRNA (miR)-140-3p was validated as a biomarker for osteoarthritis. However, the mechanism by which miRNA-140-3p regulates osteoarthritis remains unclear. Thus, this study aims to evaluate the potential function of miRNA-140-3p during the pathogenesis of osteoarthritis. MiRNA-140-3p expression in tissue and CHON-001 chondrocyte cells was determined with quantitative real time (qRT)-PCR. In vitro osteoarthritis model was established by treatment of the chondrocyte cells CHON-001 with interleukin (IL)-1β. Cell proliferation and apoptosis were measured with cell counting kit-8 (CCK8) and Annexin V/propidium iodide (PI) apoptosis assay, respectively. Protein expressions were evaluated using Western blot. The target gene of miR-140-3p was predicted using Targetscan and miRDB. MiR-140-3p was downregulated in knee tissue from patients with osteoarthritis. IL-1β inhibited the proliferation of CHON-001 cells via inducing apoptosis. In addition, IL-1β significantly inhibited the expressions of collagen II and aggrecan and increased the level of matrix metalloproteinase (MMP)13. However, the effects of IL-1β could be ameliorated by the addition of miR-140-3p mimics. Moreover, luciferase reporter assay demonstrated CXCR4 as a target gene of miR-140-3p. IL-1β-induced upregulation of CXCR4 could be blocked by miR-140-3p mimics. Our study indicated that miR-140-3p could suppress the progression of osteoarthritis by directly targeting CXCR4. Therefore, miR-140-3p might serve as a potential therapeutic target for the treatment of osteoarthritis.