Organophosphorus (OP) insecticides are widely used in agriculture, which are toxic to insect pests and nontarget organisms. The current study mainly assessed the effect of the pesticide phoxim on oxidative stress by certain biomarkers in the fat body and midgut of the silkworm, Bombyx mori (L.), after exposure to 50% lethal concentration (LC₅₀) of phoxim for 2 h. Malondialdehyde (MDA) content, activity of glutathione transferase (GST), and expression of GST at transcriptional level were assayed. LC₅₀ value of phoxim was 2.5 mg/liter at 2-h exposure for the day 3 of the fifth-instar larvae. After exposure of phoxim, MDA content in the fat body significantly increased at 4–20 h posttreatment (p.t.), the highest increase was ≈4.11-fold from 0.451 ± 0.053 to 1.854 ± 0.113 nmol/mg protein compared with corresponding control. In the midgut, significant increase in the MDA content (from 1.40- to 3.16-fold) was observed at 8–42 h p.t. The activity of GSTs increased to 1.48–2.00-fold at 24–42 h p.t. and 1.33–1.48-fold at 20–24 h p.t. in the fat body and midgut, respectively. The peroxidase activity of GSTs also was induced, which increased to 1.46–2.06-fold and 1.31–1.50-fold in the fat body and midgut, respectively. BmGSTe8 showed a late up-regulation of transcripts at 24–42 h after exposure to phoxim, which might contribute to the improved phoxim tolerance of silkworm larvae. These results indicated that phoxim could trigger oxidative stress and that MDA content and GST activity might be used as biomarkers of OP insecticide exposure. In addition, activity of GSTs were more inducible in the fat body than in midgut.