Neuroplasticity in the prefrontal cortex (PFC) after fear conditioning has been suggested to regulate the formation and expression of fear memory. Protein kinase Mζ (PKMζ), an isoform of protein kinase C with persistent activity, is involved in the formation and maintenance of memory. However, less is known about the role of PKMζ in the PFC in the formation of fear memory. We investigated whether the overexpression of PKMζ enhances the formation of auditory fear memory in rats. We found that microinfusion of lentiviral vector-expressing PKMζ into the prelimbic cortex (PrL) selectively enhanced the expression of PKMζ without influencing the expression of other isoforms of PKC. The overexpression of PKMζ in the PrL enhanced the formation of long-term fear memory without affecting short-term fear memory, whereas the overexpression of PKMζ in the infralimbic cortex had no effect on either short-term or long-term fear memory. The overexpression of PKMζ in the PrL had no effect on anxiety-like behavior or locomotor activity. We also found that PKMζ overexpression potentiated the fear conditioning-induced increase in the membrane levels of glutamate subunit 2 of AMPA receptors in the PrL. These results demonstrate that the overexpression of PKMζ in the PrL but not infralimbic cortex selectively enhanced the formation of long-term fear memory, and PKMζ in the PrL may be involved in the formation of fear memory.