Clozapine, an atypical antipsychotic agent, has been reported to cause acute hyperglycemia. However, the mechanism of clozapine-induced rapidly developing hyperglycemia is not well elucidated. To clarify the mechanism underlying clozapine-induced acute hyperglycemia, we investigated the effects of single intravenous administration of clozapine on the serum concentrations of glucose and several endogenous substances in rats. Male Wistar rats received an intravenous injection of saline (control) or clozapine 2.5, 5, 10 mg/kg. Blood samples were obtained periodically after clozapine administration to determine the serum concentrations of glucose, adrenaline, glucagon, insulin, corticosterone, and clozapine. The serum concentrations of glucose, adrenaline, and glucagon increased dose-dependently after the administration of clozapine at 2.5–10 mg/kg, and reached maxima at 5 mg/kg of clozapine. The serum concentration of corticosterone increased after the administration of clozapine, but no significant variation was observed with the dosage of clozapine. The concentration of serum insulin increased in a dose-dependent manner after clozapine administration. In conclusion, a single administration of clozapine increased the serum concentration of glucose in rats, and adrenaline and/or glucagon would be associated with clozapine-induced acute hyperglycemia.