Diabetes-induced glutathione (GSH) decrease is usually ascribed to GSH oxidation. Here we investigate, in streptozotocin-treated rats, if impairment of GSH synthesis contributes to GSH decrease in diabetic liver, and if antioxidant treatments can provide protection. Diabetic rats were divided into 3 groups: untreated diabetic rats (UD); N-acetyl-cysteine (NAC)-treated diabetic rats; taurine (TAU)-treated diabetic rats; a group of non-streptozotocin-treated rats was used as control (CTR). All rats were sacrificed at 40 weeks of age. Diabetes induced hepatic glutathione decrease, but oxidized glutathione (GSSG) did not increase significantly. Accumulations of cysteine and cysteinyl-glycine in UD suggest respectively decreased glutathione synthesis and increased loss through the plasma membrane with subsequent degradation. Decreased expression of γ-glutamyl-cysteine synthetase in UD is consistent with repressed GSH synthesis. Moreover, diabetes caused increase of GSSG/GSH ratio and induction of heme oxygenase-1, both signs of oxidative stress. Supplementation with NAC or TAU resulted in amelioration of glutathione levels, probably depending on antioxidant activity, more efficient glutathione synthesis and decreased GSH loss and degradation. In conclusion, impaired synthesis and increased loss and degradation of GSH appear to contribute to a decrease in GSH levels in diabetic liver. NAC and TAU are able to partially protect from oxidative stress and GSH decrease, while enhancing GSH synthesis and restricting GSH loss.