Objectives This study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA). Methods Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1 beta. Subsequently the expression of collagen type II alpha 1 (Col2 alpha 1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminogly-cans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-.B)antagonist MG132. The changes in Col2a1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test. Results Expression of miR-320a was elevated in OA cartilage tissues and chondrocytes, and in IL-1 beta-stimulated C28/I2 cells (p < 0.05 or p < 0.01). MiR-320a overexpression enhanced IL-1 beta-induced down-regulation of Col2a1 and ACAN and sGAG, and increased the IL-1 beta-induced overexpression of MMP-13 (p < 0.01). PBX3 was a direct target of miR-320a. PBX3 and MG132 co-transfection attenuated the effects of miR-320a on the expression of Col2a1, ACAN, sGAG and MMP-13(p < 0.01). Conclusion Overexpression of miR-320a might enhance IL-1 beta-induced cartilage degradation factors. These effects might be via targeting PBX3 and regulating NF-kappa B.