In this study, the effect of ocean acidification on sex differentation of marine medaka was studied. Fertilized eggs were incubated at three concentrations of CO₂ (480×10^-6, 1 000×10^-6 and 2 000×10^-6) and after hatching, the fry were bred under control condition (480×10^-6) until adulthood. Phenotypic sex was judged from secondary sexual characteristics. The ratio of male to female in two treatment groups was significantly higher than that in the control group (P<0.01). Genotypic sex was determined by the presence or absence of the DMY gene. In the control group, phenotypic sex completely agreed with genotypic sex. However, in two treatment groups, there are some phenotypical males but genotypic female, which is sex-reversed XX male. The sex reversal rate was 38.4% and 47.5% at 1 000×10^-6 and 2 000×10^-6 concentration of CO₂, respectively.