Nonylphenol (NP) is a representative environmental endocrine-disrupting compound that can induce oxidative stress in organisms. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway acts an important role in preventing oxidative stress. The aim of this study was to investigate the influence of oxidative stress caused by NP on Keap1-Nrf2 pathway in rats. Rats were treated with NP (30, 90, 270 mg/kg) for different exposure time (7, 14 and 28 days). The levels of reactive oxygen species (ROS) in serum and glutathione S-transferase (GST), UDP-Glucuronosyl Transferase (UGT) in liver were detected by ELISA kits. Western blot was used to detect Keap1, Nrf2 protein expression in liver and cerebral cortex. The results showed that 28 days of NP exposure significantly increased ROS levels in NPH group. And 14 days exposure to NP significantly enhanced the levels of GST and UGT, while 28 days of exposure showed a suppressive effect. In liver, Keap1 levels was upregulated at 7, 14 and 28 days of NP exposure, while nuclear Nrf2 levels decreased at 7 and 28 days but increased at 14 days. In cerebral cortex, Keap1 and Nrf2 expression increased at 14 days but decreased at 28 days. Besides, with the prolongation of NP exposure time, the GST and UGT levels in NPM and NPH groups were increased firstly and then decreased, while Keap1 and Nrf2 protein levels were constantly decreased in liver and cerebral cortex. In conclusion, the lower dose and shorter exposure time of NP activated the Keap1-Nrf2 pathway that may reduce the damage of oxidative stress, but when further exposed to NP at higher dose and time, the pathway could be inhibited.