Objective To evaluate the protective efficacy of gossypin (3,3',4',5,7,8-hexahydroxyflavone 8-glucoside) by administering it intraperitoneally, for dose, time, and vehicle dependent effects against sulphur mustard (SM), administered through percutaneous route in mice. Methods SM (diluted in PEG-300) was administered percutaneously. The protective efficacy of gossypin was evaluated by administering it intraperitoneally (50, 100, 200, and 400 mg/kg), in various vehicles (water, PEG-300 and DMSO), and time intervals (30 min prior, simultaneous and 2 h post). The time dependent protection of gossypin (200 mg/kg in PEG-300; i.p.) was also evaluated using selected biochemical variables (GSH, GSSG MDA, total antioxidant status, Hb, WBC count, RBC count, glutathione peroxidase, glutathione reductase, and superoxide dismutase) and liver histology. The protection of gossypin by oral route was also evaluated against percutaneously administered SM. Results The protection against systemic toxicity of SM (LD50 8.1 mg/kg) was better when gossypin was given with PEG-300 (8.0 folds) than DMSO (5.7 folds). No protection was observed when gossypin was administered with water. Good protection (8.0 folds) was observed when gossypin was administered (200 mg/kg in PEG-300; i.p.) at 30 min prior or simultaneous to SM exposure, but no protection was observed when gossypin was administered 2 h post to SM exposure. A significant weight loss was observed 7 days after SM administration (2 LD50), with a significant increase in RBC and Hb. A significant decrease in total antioxidant status of plasma, liver GSH and GSSG levels, and in the activities of glutathione peroxidase, glutathione reductase and superoxide dismutase was also observed 7 days after SM administration. SM treated mouse liver also showed necrosis. A significant protection was observed when gossypin (200 mg/kg in PEG-300; i.p.) was administered either as a pretreatment (30 min before) or simultaneous treatment, and not as a post treatment (2 h). The protective efficacy of gossypin was better through oral route when administered with DMSO (4.8 folds) than with PEG-300 (2.4 folds). No protection was observed when gossypin was administered orally with water. Conclusion Percutaneous administration of SM induces oxidative stress and gossypin can protect it as a prophylactic agent by intraperitoneal or oral routes. Copyright © 2007 by China CDC.