Isoniazid (INH), one of the first-line antituberculosis drugs, has potential liver toxicity. Mechanisms reported by previous studies mainly focused on oxidative stress. In the present study, we investigated acute effects of diallylsulfide (DAS), a selective CYP2E1 inhibitor, on reduced glutathione (GSH) and reactive oxygen species (ROS) levels in rat primary hepatocytes treated with INH. In cultures treated with INH for 1, 4, 8h, significant loss of GSH content and decrease of ROS levels were observed. Moreover, when hepatocytes were co-treated with INH and 1mM DAS, accelerated GSH depletion and increased ROS production appeared. Further more, rat primary hepatocytes survival rates decreased significantly in cultures treated with INH together with DAS than in cultures treated with INH alone after 24h. In conclusion, DAS could potentiate INH toxic effect and this is the first study reporting the effect of DAS on oxidative stress in INH-induced hepatocytotoxicity.