High relative humidity is associated with high mortality in insects due to the infection with entomopathogens. However, the mechanism is still not fully understood. In this study, bioassay for Spodoptera exigua nucleopolhedrovirus (SeNPV) and antioxidant enzyme activity, total antioxidant capacity, and hydroxyl radical scavenging capacity were measured in Spodoptera exigua (Hübner) larvae under three relative humidities (RH) conditions (60–70 %, 75–85 %, and 90–100 %). Results showed that humidity significantly affected the toxicity of SeNPV against S. exigua larvae, and the LD₅₀ (lethal dose 50 %) of SeNPV against S. exigua larvae was 1539.8 (60–70 % RH), 1110.4 (75–85 % RH) and 567.6 PIBs/g (90–100 % RH). The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) activities of S. exigua larvae were significantly lower in those subjected to high humidity than those in the control humidity group (60–70 % RH). The capacity of total antioxidant and hydroxyl radical scavenging decreased with the increasing of relative humidity. These results suggest that high RH may cause a decrease in the antioxidant system of S. exigua larvae, which may explain high RH is always associated with high mortality in insects due to infection with pathogens.