In this study, effects of sublethal concentrations of methidathion on catalase (CAT), superoxide dismutase (SOD), glutathion peroxidase (GSH-Px) activities and lipid peroxidation with expression of heat shock protein (HSP70) gene in liver tissues of Oreochromis niloticus were investigated. The 96-h LC50 value for methidathion was determined as 0.1045 mg/L for O. niloticus in this study. 0.013 (1/8 of LC50) and 0.026 mg/L (1/4 of LC50) sublethal concentrations were applied for 4 and 10 days in this experiment. CAT and GSH-Px activities in liver tissue were increased, in relation to both the time and doses applied. SOD activities were increased under the influence of both concentrations at the 4th day, but on 10th day, it was decreased with influence of 0.026 mg/L methidathion concentration. The tissue MDA levels were significantly increased in relation to both the time and dose applied. The relative mRNA levels of the HSP70 gene were detected by real time polymerase chain reaction (RT-PCR). The mRNA levels of HSP70 gene by methidathion increased significantly on 4th days compared to control, but decreased significantly on 10th days.