Despite the importance of prefrontal cortical dopamine in modulating reward, little is known about the implication of the specific subregion of prefrontal cortex in opioid reward. We investigated the role of neurons projecting from the ventral tegmental area (VTA) to the anterior cingulate cortex (ACG) in opioid reward. Microinjection of the retrograde tracer fluorogold (FG) into the ACG revealed several retrogradely labelled cells in the VTA. The FG-positive reactions were noted in both tyrosine hydroxylase (TH)-positive and -negative VTA neurons. The released levels of dopamine and its major metabolites in the ACG were increased by either the electrical stimulation of VTA neurons or microinjection of a selective μ-opioid receptor (MOR) agonist, (D-Ala²,N-MePhe⁴,Gly-ol⁵) enkephalin (DAMGO), into the VTA. MOR-like immunoreactivity was seen in both TH-positive and -negative VTA neurons projecting to the ACG. The conditioned place preference induced by intra-VTA injection of DAMGO was significantly attenuated by chemical lesion of dopaminergic terminals in the ACG. The depletion of dopamine in the ACG induced early extinction of μ-opioid-induced place preference. The levels of phosphorylated DARPP32 (Thr34) and phosphorylated CREB (Ser133) were increased in the ACG of rats that had maintained the morphine-induced place preference, whereas the increases of these levels induced by morphine were blocked by pre-treatment of a selective dopamine D1 receptor antagonist SCH23390. These findings suggest that VTA-ACG transmission may play a crucial role in the acquisition and maintenance of μ-opioid-induced place preference. The activation of DARPP32 and CREB through dopamine D1 receptors in the ACG could be implicated in the maintenance of μ-opioid-induced place preference.