Kappa-opioid receptors (KORs) are mediators of motivational processes, mood/emotion, and stress reactivity. KORs are enriched in brain regions that mediate such behaviors, including the medial prefrontal cortex (mPFC). The mPFC guides behavior through its connections with limbic brain regions such as the amygdala, hippocampus, and ventral tegmental area. A critical knowledge gap exists in our understanding of the role of KOR systems in modulating mPFC afferents. Thus, we designed a series of studies to understand the role of KORs in regulating dopamine (DA) and limbic glutamatergic afferents to the mPFC. First, we determined the role of KOR systems in regulating neurotransmission in the mesocortical DA pathway by utilizing a combination of neurochemical techniques in rats and in transgenic mice. We found that mPFC KOR activation and antagonism decreased and increased DA output, respectively. Genetic ablation of KOR in DA neurons abolished the inhibitory effect of mPFC KOR signaling on local DA output. These findings suggest that mPFC KORs negatively modulate the mesocortical DA pathway by directly acting on DA varicosities in the mPFC. Second, we examined the role of mPFC KOR systems in regulating mPFC extracellular glutamate and glutamatergic limbic inputs utilizing neurochemical and electrophysiological techniques in rats. We found that extracellular glutamate and glutamate-driven GABA levels were inhibited by mPFC KORs. mPFC KOR activation inhibited synaptic transmission in the BLA to mPFC pathway in a manner not overcome by BLA burst stimulation. KORs do not inhibit the hippocampus to mPFC pathway. These findings show that mPFC KORs inhibit glutamatergic afferents in a pathway-specific manner. Lastly, we characterized heterosynaptic interactions between the BLA and hippocampus in the mPFC, and determined the role of KORs in these interactions. BLA train stimulation inhibits hippocampal inputs to the mPFC in an activity-dependent manner, while hippocampus stimulation is without effect on BLA-evoked responses. KOR antagonism does not modify BLA-evoked heterosynaptic suppression, suggesting that BLA-evoked heterosynaptic suppression is KOR-independent. This series of studies demonstrate that mPFC KORs regulate mesocortical DA and limbic pathways, but may not be involved in limbic interactions. These findings provide a biological framework whereby mPFC KOR signaling alters mPFC-dependent behaviors.