Objective To investigate the mechanism underlying the effect of intra-articular injection of ozonated water on cartilage repair in knee osteoarthritis (KOA) rats via the miR-214-3p/nuclear factor (NF)-κB signaling pathway, to provide a theoretical basis for the clinical application of ozonated water. Methods Sixty male Sprague-Dawley rats were divided randomly into ozonated and experimental groups. Rats in the experimental group were further divided randomly into model, ozonated water, ozonated water + AAV-NC, and adeno-associated virus with ozonated water+AAV-miR-214-3p inhibitor groups. The articular cartilage repair status was evaluated after the respective treatments, using articular cartilage surface gross scoring, hematoxylin/eosin staining, and modified Mankin’s score. Expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and matrix metalloproteinase (MMP)-13 were detected by enzyme-linked immunosorbent assay (ELISA). Protein expression levels of IκB kinase (IKK) β, p65, and phospho (p)-IκBα were detected in cartilage tissues by Western blot, and gene expression levels of IKKβ, p65, IκBα, and miR-214-3p were detected by real-time polymerase chain reaction (RT-PCR). Results Rats in all groups showed different degrees of cartilage damage compared with the control group, and the scores were significantly lower in the ozonated water and ozonated water+AAV-NC groups compared with the ozonated water+AAV-miR-214-3p inhibitor group (P<0.05). IL-1β, TNF-α, and MMP-13 levels detected by ELISA were significantly lower in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups (P<0.05). According to RT PCR, IKKβ and p65 mRNA levels were significantly down-regulated while IκBα mRNA was significantly up-regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups (P<0.05). Similarly, IKKβ, p65, and p-IκBα protein expression levels detected by Western blot were significantly down-regulated while IκBα protein expression levels were up regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups (P<0.05). Conclusions Ozone hydrotherapy significantly ameliorated cartilage damage in KOA rats, possibly via inhibiting activation of the NF-κB signaling pathway through up-regulation of miR-214-3p expression and promoting cartilage repair in KOA rats.