Many studies in humans and rodents have shown that the anterior cingulate cortex (ACC) plays a critical role in the regulation of pain-related aversion and that the projection from the rostral ACC (rACC) to the ventral tegmental area (VTA) is implicated in this modulation process. The ACC is also reported to be involved in the regulation of itch-scratch behavior. However, it remains unclear whether the ACC is involved in the modulation of the negative emotions induced by acute itch sensation. In this study, we investigated the pruritogen-induced conditioned place aversion (CPA) and itch-scratching behavior in rats after pharmacogenetic inhibition of the activities of rACC-VTA pathway or the rACC neurons, respectively. Pharmacogenetic inhibition of glutamatergic neurons of rACC projecting to the VTA alleviated the CPA responses and itch-scratching behavior induced by the subcutaneous injection of 5-HT, a nonhistamine-dependent pruritogen. However, pharmacogenetic inhibition of rACC neurons did not change the CPA behavior associated with itch and, conversely, increased itch-scratching behavior. These results reveal that a specific subpopulation of rACC neurons projecting to the VTA positively regulates itch sensation and the negative emotion accompanying itch, whereas the global rACC negatively modulates acute non-histaminergic itch in rats. Postsynaptic GABAergic neurons in the VTA may mediate emotion modulation of the rACC-VTA pathway. The current findings contribute to a better understanding of the circuit mechanisms underlying the processing of different components of itch, such as sensation and emotion.