Formerly considered as an exclusively peripheral receptor, it is now accepted that CB2 cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB2 receptors in the brain need to be clarified. The aim of our work was to study the μ-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB2 receptor antagonist SR144528 in brainstem of mice deficient in either CB1 or CB2 receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB2 cannabinoid antagonist SR144528, suggesting a CB2 receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [35S]GTPγS binding assay to analyze the capability of μ-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB1 wild-type and CB1 knockout mice after a single injection of SR144528 at 0.1 mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB1 wild-type and CB1 knockout mice. In vitro addition of 1 μM SR144528 caused a decrease in the maximal stimulation of DAMGO in [35S]GTPγS binding assays in CB2 wild-type brainstem membranes whereas no significant changes were observed in CB2 receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.