Although the widespread use of the oxygen-ozone in pain management, there is currently no consensus on its mechanisms of action and nearly no report for its action on nervous cells. Accordingly, the present study was designed to assess the effects of oxygen-ozone on astrocytes. Astrocytes were cultured in vitro through methods of trypsinization, different-speed cultivation and passaging to purify, then seeded into 24 well plates and divided to one of four groups (n=7) to receive the following treatments: respectively added 400 microl complete medium (CM) after effects of 20 microg/ml oxygen-ozone (Group O-20), 40 microg/ml oxygen-ozone (Group O-40), 60 microg/ml oxygen-ozone (Group O-60); without intervention (Group C). After incubation of 2 h or 4 h, cell morphology was observed and endocellular superoxide dismutase (SOD), endocellular malondialdehyde (MDA), lactate dehydrogenase (LDH) leaking ratio, and dead cells' percentage were detected. The results showed cell damage in Group O-60. As compared with Group C, endocellular SOD increased in all groups, MDA at 2 h increased in Groups O-40 and O-60 and MDA at 4 h decreased in Groups O-20 and O-40; LDH leaking ratio at 2 h in Group O-20 and those at 2 and 4 h in Group O-40 decreased, while LDH leaking ratio at 4 h increased and dead cells' percentage in Group O-60 increased. We conclude that in short time (2 and 4 h), oxygen-ozone of 60 microg/ml showed a damaging role on astrocytes in vitro, while oxygen-ozone of 20 and 40 microg/ml did not show damaging role obviously.