β-Naphtoflavone (β-NF) is a flavonoid and enhances oxidative stress in vertebrates with little information from aquatic invertebrates as yet. In this study, we investigated the effects of β-NF on the antioxidant defense systems of the intertidal copepod Tigriopus japonicus. To measure the β-NF-triggered changes in oxidative stress markers, such as intracellular reactive oxygen species (ROS), glutathione (GSH) concentration, residual glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) activity, T. japonicus were exposed to β-NF (0.5 and 1 mg/L) for 72 h. Significant (P < 0.05) induction of the intracellular ROS content (%) was observed in 1 mg/L of β-NF exposed T. japonicus, compared to the negative control and H2O2-exposed group. The GSH levels were significantly increased in the 0.5 mg/L of β-NF-exposed group for 12 h and 1 mg/L of β-NF-exposed groups for 12–24 h. GPx, GST, and GR activities showed a significant increase in the 1 mg/L β-NF-exposed group, indicating that β-NF induces oxidative stress in T. japonicus. To understand the effects of β-NF at the level of transcript expression, a 6K microarray analysis was employed. Transcript profiles of selected antioxidant-related genes were modulated after 72 h exposure to 1 mg/L of β-NF. From microarray data, 10 GST isoforms, GR, GPx, PH-GPx, and Se-GPx were chosen for a time-course test by real-time RT-PCR. T. japonicus GST-S, GST-O, GST-M, and GST-D1 were significantly increased in a 1 mg/L β-NF-exposed group. T. japonicus GPx, GR, and Se-GPx mRNA levels were also significantly increased at both concentrations. Our results revealed that oxidative stress was induced by β-NF exposure in T. japonicus. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 332–342, 2015.