Clozapine (CLZ) is an atypical antipsychotic drug used for treatment-resistant schizophrenia. The target receptors for CLZ, including dopamine D4 and serotonin 5-HT2A receptors, are expressed in various types of neurons with different expression levels. Although cellular and molecular responses by CLZ have been characterized by various studies, neuronal circuit mechanisms underlying the effect of CLZ remain elusive. Thus, it is important to identify characteristics of neurons functionally responsible for the effect of CLZ. For this purpose, we investigated anatomical features of CLZ-responsive neurons in the medial prefrontal cortex (mPFC) by genetic labeling of CLZ-activated neurons and their axons. We found that CLZ-activated neurons in the mPFC have more projections to the mediodorsal thalamus (MD) and the ventromedial thalamus than the basolateral amygdala, whereas vehicle-activated neurons in the mPFC have similar extent of projections to these three regions. We also found that the MD-projecting neurons in the mPFC were activated by CLZ not only in wild-type mice but in a mouse model of schizophrenia by retrograde tracing and immunohistochemistry for c-Fos. These results suggest that the activation of the mPFC-MD circuit is involved, at least partly, in the mechanism for the therapeutic action of CLZ.