There are many reports that show the teratogenic effects of phenobarbital can be decreased by stimulation of maternal immune system. Therefore, in this study, the prophylactic effects of levamisole and vitamin E on teratogenic effects of phenobarbital were compared. This study was performed on 20 pregnant rats that were divided into four groups. Control group received normal saline and test groups received phenobarbital (120 mg/kg), phenobarbital (120 mg/kg) plus levamisole (10 mg/kg) and phenobarbital (120 mg/kg) plus vitamin E (100 mg/kg) intraperitoneally at 9-11th days of gestation, respectively. Fetuses were collected at 20th day of gestation and after determination of weight and length; they were stained by Alizarin red - Alcian blue method. Cleft palate and spina bifida incidence were 66.66% and 69.44% in fetuses of rats that had received only phenobarbital. Cleft palate and spina bifida incidence were 65.45% and 38.18% had in the group which had received phenobarbital plus levamisole. However, Cleft palate and spina bifida incidence were 54.54% and 27.27% in the group which had received phenobarbital plus vitamin E. The arithmetic means of the weight and length of fetuses the rats that had received levamisole and vitamin E were significantly greater than that of those that had received only phenobarbital. Vitamin E had a greater prophylactic effect than levamisole on the incidence of phenobarbital-induced cleft palate and spina bifida. However, the difference was not significant.