Our understanding of the effects of experience on brain function has advanced in recent years with the realization that a variety of epigenetic processes regulate gene expression underlying learning and memory. Inhibitor of growth family member 1 (ING1) is a key epigenetic regulator, which has been identified as a reader protein for the DNA modification, 5-formylcytosine (5fC). 5fC has recently been shown to be a stable epigenetic mark; however, it is not known whether the accumulation of 5fC is functionally relevant in the adult brain. The importance of ING1 in learning and memory also remains to be demonstrated. Here, using lentiviral-mediate gene transfer, we show that ING1 activity in the medial prefrontal cortex (mPFC) is critically involved in fear extinction memory in mice. To investigate the mechanism underlying regulation of extinction memory by ING1, we have developed a novel fluorescence-activated cell sorting (FACS)-based method to enrich for neurons that have been selectively activated by learning. We applied this method to dissociate cells from the mPFC of mice immediately following extinction training, then performed ING1 ChIP-seq on protein-DNA complexes derived from those activated neurons. In this way, we targeted ING1 binding exclusively in neurons that are involved in the formation of the memory trace, and elucidate genes that are directly involved with extinction memory. Most of the targets identified have been demonstrated to be involved in various brain functions, but not previously been implicated in fear extinction. Importantly, we have discovered that some, not all, ING1 binding sites are associated with the accumulation of 5fC during extinction learning. These finding represent a potentially new mechanism of gene regulation by ING1-mediated recognition of 5fC in the adult brain, and suggest an important role for ING1 in the formation of extinction memory.