Kappa-opioid receptors (KORs) are important for motivation and other medial prefrontal cortex (mPFC)-dependent behaviors. Although KORs are present in the mPFC, their role in regulating transmission in this brain region and their contribution to KOR-mediated aversion are not known. Using in vivo microdialysis in rats and mice, we demonstrate that intra-mPFC administration of the selective KOR agonist U69,593 decreased local dopamine (DA) overflow, while reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino-acid levels were also affected by KORs, as U69,593 reduced glutamate and GABA levels driven by the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of glutamatergic mini EPSPs. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino-acid neurotransmission, and are necessary for KOR-mediated aversion.