Neocortical projection neurons consist of intracortical connected upper layer (UL, layer II–IV) neurons and subcortical connected lower layer (LL, layer V–VI) neurons. Afferent activity from the thalamus regulates layer-specific gene expression during postnatal development, which is critical for the formation of proper neocortical cytoarchitecture. Here, we show that activity-dependent gene regulation is confined to UL cortical neurons, but not LL neurons, and that this distinction is likely due to epigenetic modifications of chromatin. We found that the immediate early genes (IEGs), EGR1 and c-FOS, are downregulated in all cortical laminar layers in the absence of afferent activity in vivo. Transcriptional assays demonstrated that EGR1 and c-FOS are able to bind to the promoters of UL- and LL-specific genes to induce transcription. Furthermore, we discovered that LL neurons express higher levels of heterochromatin markers, such as H3K9m3 and H4K20m3, compared to UL neurons. Our results suggest that differential epigenetic modifications of chromatin is an intrinsic mechanism that underlies the different sensitivities of cortical neurons to activity-dependent gene regulation.