Agmatine, an endogenous ligand for imidazoline receptor, has been shown to prevent opioid dependence, but not much is known about the mechanisms of the effect of agmatine. In the present study, we investigated the function of I1 imidazoline receptor and its candidate protein imidazoline receptor antisera-selected (IRAS)/Nischarin in morphine dependence as well as in the effect of agmatine inhibiting morphine dependence by pharmacological and molecular approaches. Results showed that inhibition of IRAS or Nischarin did not change the development of morphine dependence in vitro and in vivo under the basal condition. Agmatine could reduce the cyclic 3′, 5′ adenosine monophosphate (cAMP) overshoot at the concentration of 0.01–10 µM in the primary cultured rat hippocampal neurons and attenuated the withdrawal signals and the elevation of FosB and ΔFosB at the dose of 5 mg/kg in the morphine-dependent mice. The effect of agmatine was inhibited by efaroxan (I1 imidazoline receptor non-specific antagonist) and the RNA interference against IRAS or Nischarin. These findings indicate that I1 imidazoline receptor or IRAS/Nischarin mediates the effect of agmatine on morphine dependence and provide evidence that I1 imidazoline receptor may be a new target for treating morphine dependence.