In this study, thiol/disulfide homeostasis in the liver tissues of high-fructose-fed rats was investigated in conjunction with the changes in the main hepatic detoxification enzyme, glutathione S-transferase (GST). Additionally, the effects of well-known probiotics namely Kefir, Lactobacillus helveticus, and Lactobacillus plantarum supplementation on the thiol/disulfate contents and GST activity and gene expression levels were analyzed. Fructose, administered as a 20% solution in drinking water for 15 weeks, developed an animal model of metabolic syndrome in male Wistar rats. Kefir, L. helveticus, and L. plantarum supplementations were given by gastric gavage once a day during the final 6-weeks. The changes in hepatic GST were determined with kinetic-optimized spectrophotometric enzyme assays and qRT-PCR. Total thiol, native thiol, and disulfide levels were analyzed using (5,5-dithio-bis-(2-nitrobenzoic acid) as a chromogenic agent. High-fructose consumption reduced total and native thiol contents while increasing disulfide levels in the liver tissues of rats. Kefir and L. plantarum normalized the thiol levels and all probiotics reduced disulfide contents. High fructose augmented total GST activity but reduced the GST-Mu isoform. L. helveticus and L. plantarum normalized the total and GST-Mu activity, respectively. These results demonstrated a shift toward disulfide formation in the hepatic tissues of rats fed with high fructose. A possible reason would be the increase in total GST activity that uses the free glutathione, the main native thiol source in cells, as a substrate. Besides, probiotics such as Kefir, L. helveticus, and L. plantarum have an improving effect on thiol/disulfide homeostasis as well as main detoxification enzymes.