Objective To investigate the roles of and relationship between microRNA (miR)-330-3p and S100 calcium-binding protein B (S100B) in an animal model of cartilage injury. Methods This study included 30 New Zealand male rabbits randomly divided into three groups: an intervention group, a model group and a sham surgery control group. Modelling was performed in the intervention and model groups, but in the sham surgery group, only the skin was cut. After modelling, the intervention and model groups were injected with the miR-330-3p overexpression vector GV268-miR-330-3p or the control GV268-N-ODN vector, respectively, twice a week for 7 weeks. Results Levels of interleukin-1β and tumour necrosis factor-α in the synovial fluid were significantly higher in the model group than in the intervention and control groups. The level of miR-330-3p in the cartilage tissue was significantly higher in the control group than in the model group but it was significantly lower compared with the intervention group. Levels of S100B, fibroblast growth factor receptor 1 and fibroblast growth factor-2 in the cartilage tissue of rabbits in the model group were significantly higher compared with the control and intervention groups. Conclusion These findings demonstrate that the upregulation of miR-330-3p can inhibit the expression of S100B.