Osteoarthritis (OA) is a degenerative disease characterized by deterioration of articular cartilage. The aim of this study was to identify and characterize the expression of microRNA-125b-5p (miR-125b-5p) in normal and OA synovial cells, and to determine its role in OA pathogenesis. First, the levels of miR-125b-5p and synoviolin 1 (SYVN1) were detected among normal, mild OA and severe OA groups with the use of quantitative PCR. Computational analysis was used to search for the target of the miR-125b-5p, and luciferase reporter assay system was used to validate SYVN1 as the target gene of miR-125b-5p. Then the SYVN1 expression level of cells transfected with miR-125b-5p mimics or inhibitors was estimated using quantitative PCR and western blotting. Finally, MTT assay was employed to estimate the effect of miR-125b-5p on apoptosis. We enrolled 36 participants consisting of 12 normal control, 12 mild OA and 12 severe OA, furthermore, we performed quantitative PCR to detect the levels of miR-125b-5p and SYVN1 among those groups, and found that miR-125b-5p was expressed at highest level in severe OA compared with normal control and mild OA groups, while SYVN1 was expressed at the lowest level in severe OA. Additionally, we identified that SYVN1 is a target of miR-125b-5p by using computational analysis and luciferase assay. Transfection with miR-125b-5p mimic or inhibitor was employed to investigate the effect of miR-125b-5p on expression of SYVN1 in synovial cells, and synovial cell viability and apoptosis, and the results showed that miR-125b-5p mimics significant decreased the expression of SYVN1, a substantially promoted apoptosis of synovial cells, while miR-125b-5p inhibitors remarkably increased the level of SYVN1, and substantially suppressed apoptosis of synovial cells. The data suggested that miR-125b-5p promoted apoptosis of synovial cells through targeting SYVN1 gene, with important implication for validating miR-125b-5p as a potential target for OA therapy.