The present study was aimed to investigate the metabolomics of sulfur amino acids in Zucker diabetic fatty (ZDF) rats, an obese type 2 diabetic animal model. Plasma levels of total cysteine, homocysteine and methionine, but not glutathione (GSH) were markedly decreased in ZDF rats. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine and spermine were also decreased. There are no significant difference in hepatic S-adenosylmethionine, S-adenosylhomocysteine, GSH, GSH disulfide, hypotaurine and putrescine between control and ZDF rats. Hepatic SAH hydrolase, betaine-homocysteine methyltransferase and methylene tetrahydrofolate reductase were up-regulated while activities of gamma-glutamylcysteine ligase and methionine synthase were decreased. The area under the curve (AUC) of methionine and methionine-d4 was not significantly different in control and ZDF rats treated with a mixture of methionine (60mg/kg) and methionine-d4 (20mg/kg). Moreover, the AUC of the increase in plasma total homocysteine was comparable between two groups, although the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine after the methionine loading was rapidly increased and normalized in ZDF rats. These results show that the AUC of plasma homocysteine is not responsive to the up-regulation of hepatic BHMT in ZDF rats. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels.