Association between elevated plasma homocysteine levels and insulin resistance has been reported, however, whether hyperhomocysteinemia induces insulin resistance or it is actually hyperinsulinemia that causes elevated plasma homocysteine levels, the direction of causality in this association is not still clear. In this study, we examined the hypothesis that hyperhomocysteinemia may cause hyperinsulinemia leading to insulin resistance in rats. Plasma glucose, insulin and total homocysteine concentrations were determined in two groups of male Sprague-Dawley rats, a test group that administered with homocysteine and a control group with no homocysteine in daily drinking water before and after 50 days. Oral glucose tolerance tests were also performed in control and test groups before and after 50 days. Mean fasting plasma insulin level was significantly higher (42.5+/-20.4 mU/L versus 23.2+/-5.9 mU/L, p=0.01), whereas mean glucose: insulin ratio was significantly lower in test rats than in control rats (0.12+/-0.07 versus 0.17+/-0.05, p=0.04) after 50 days. In addition, mean homeostasis assessment insulin resistance index was significantly higher in test rats than in control rats (7.5+/-3.5 versus 4.0+/-1.6, p=0.02) after 50 days. The mean plasma glucose level was not significantly different (4.1+/-1.1 mmol/L versus 3.9+/-0.8 mmol/L, p=0.57) between controls and test rats, however, the results from oral glucose tolerance tests showed the development of insulin resistance in test rats after 50 days administration of homocysteine. Results from this in vivo study suggest that homocysteine can cause insulin resistance and this relationship may need to be considered when evaluating the role of plasma homocysteine as a risk factor in patients with obesity and type II diabetes.