The present study investigated the effect of agmatine on acquisition and expression of ethanol conditioned place preference (CPP) and its modulation by imidazoline agents. Swiss albino mice were treated intraperitoneally with saline or agmatine (20–40 mg/kg) before injection of ethanol (1.25 mg/kg) during conditioning days or on a test day (20–120 mg/kg), to observe the effect on acquisition or expression of CPP, respectively. Agmatine inhibited the acquisition but not the expression of ethanol CPP. Furthermore, both the I1 receptor antagonist, efaroxan (9 mg/kg) and the I2 receptor antagonist, BU224 (5 mg/kg) attenuated the agmatine-induced inhibition of the ethanol CPP acquisition. In contrast, the I2 receptor agonist, 2-BFI (5 mg/kg) and I1 receptor agonist, moxonidine (0.4 mg/kg) alone, or a combination of their subeffective doses, significantly attenuated the effect of agmatine (20 mg/kg) on acquisition of ethanol CPP. Agmatine or imidazoline agents alone produced neither place preference nor aversion, and at the doses used in the present study did not affect locomotor activity. Thus, agmatine attenuates the acquisition of ethanol CPP at least in part by imidazoline (I1 or I2) receptors. In future studies, agmatine or agents acting at the imidazoline receptors could be explored for their therapeutic potential in ethanol dependence.