Objective To observe the effects of different concentrations of medical ozone(O3) on damaged articular cartilage cells induced by interlukin-1β(IL-1β), and discuss the rational concentration boundry of O3 in the clinical practice. Methods Cartilaginous layers of knee were separated from seven-month old NewZealand rabbit under semi-asepsis environment and then digested under sterile environment to get the cartilage cells. The cartilage cells were cultured and subcultured after digested under sterile environment. The experimental cells were the third generation cartilage cells. Chondrocytes were divided randomly into eight groups cultured with different mediums: the normal chondrocytes group, chondrocytes induced by IL-1βas the model cell group, chondrocytes with O3(20, 40, 60μg/mL) as the control groups, chondrocytes induced by IL-1β with O3(20, 40, 60μg/mL) as the experimental groups. After 24 hours, the cells morphology was observed. Malondialdehyde (MDA), tumor necrosis factor-α (TNF-α)and matrix metalloproteinase-3 (MMP-3) content in the cartilage cell culture fluid were detected by enzyme-linked immunosorbent assay (ELISA). Results The shape of the cells in the experimental group, the control group and the normal cartilage cells group was comparable, after induced by 20, 40μg/mL concentrations of O3. A few cartilage cells induced by low and intermediate concentration of O3 showed pyknosis, and the levels of MDA, TNF-αand MMP-3 in the cartilage cell culture fluid were significantly lower than those of the model group (P<0.05). Cartilage cells induced by 60μg/mL concentration of O3, were seriously damaged, even showed apoptosis. MDA, TNF -αand MMP-3 content in the cartilage cell culture fluid was significantly higher than that of the model group (P<0.05). Conclusion The groups of 20, 40μg/mL concentration of O3 have a protective effect by lowing concentration of inflammatory factors significantly in chondrocytes of osteoarthritis. The 60μg/mL concentration of O3 damages either the cartilage cells in the pathological process of osteoarthritis or normal cartilage cells.