Guanidine is an active ingredient extracted from Galega officinalis. It is considered as ligand for imidazoline receptor due to the similarity of chemical structure. Previous studies showed that an activation of imidazoline I2 receptor (I2R) in adrenal gland lowered plasma glucose through releasing beta-endorphin to stimulate the opioid-mu receptor in streptozotocin-induced diabetic rats (STZ rats). However, the effect of guanidine on I2R remains unclear. In the present study, we observed that guanidine decreased plasma glucose in STZ rats, which was blocked by I2R antagonist (BU224) but not by opioid receptor antagonist (naloxone) or opioid-mu receptor antagonist (naloxonazine). The stimulatory effect of guanidine on glucose uptake in skeletal muscle from Wistar rats was observed and this effect was also abolished by BU224. Then, we used amiloride, an established blocker of I(2A), to differentiate the subtype of I2R for this action of guanidine. Results show that guanidine-induced glucose uptake into skeletal muscle was not blocked by amiloride except at concentrations higher than 2microM showing the mediation of I(2B)R. Taken together, the decrease of plasma glucose by guanidine seems not related to release of endogenous opioid to activate opioid-mu receptor but through direct activation of imidazoline I2R while the I(2B) subtype is responsible for increase of glucose uptake into skeletal muscle. Thus, guanidine can be applied as the ligand or agonist of imidazoline I(2B) receptor.