Pyramidal neurons located within layer V of the medial prefrontal cortex drive cognitive circuits by integrating afferent signals and sending efferent projections to cortical and subcortical targets. This role is supported by cholinergic neurotransmission, which modulates pyramidal neuron excitability via postsynaptic nicotinic receptors. We employed whole-cell electrophysiology with neuron reconstruction in brain slices from mice of both sexes to demonstrate that medial prefrontal layer V pyramidal neurons comprise three subtypes that have distinct electrophysiological properties, receptor isoform-specific nicotinic responses, and projection targets. Burst-firing neurons may be sub-divided into subtypes having (i) α7 isoform nicotinic responses and projections to the contralateral cortex, or (ii) α7 and β2 isoform nicotinic responses and projections to the nucleus accumbens. Regular-firing neurons have β2 isoform nicotinic responses and projections to the ventromedial thalamus. These findings provide new insight into an isoform-specific mechanism by which cholinergic neurotransmission modulates distinct efferent projections from this cognitive brain region.