Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating. PPI deficits have been associated with a number of neuropsychiatric disorders, including schizophrenia. Differential PPI has been demonstrated also across various inbred mouse strains; however, the molecular mechanisms underlying these differences in sensorimotor gating remain unclear. Here, we sought to identify gene expression in the medial prefrontal cortex (mPFC) of mice associated with PPI using a laser microdissection and microarray analysis-based approach. C57BL/6 mouse substrains were used for the study as they have dramatically different PPI. Transcriptional analysis of closely related substrains was predicted to reduce the detection of genetic variation incidental to the phenotype. Microarray analysis comparing the mPFC of C57BL/6J to C57BL/6NHsd mice revealed neurotransmission- and cellular stress-related transcriptional responses associated with lower PPI. Down-regulation of metabotropic glutamate receptor 5, phospholipase C, and inositol monophosphatase 1 gene expression suggest altered phosphoinositide signaling, while decreased expression of a gamma-amino-butyric acid (GABA)A receptor subunit implies changes in GABAergic signaling. Genes involved in neuronal excitation and protection were also differentially expressed, including up-regulation of five immediate early genes and anti-apoptotic/survival factors as Bcl2-associated athanogene 3 and brain-derived neurotrophic factor. These data support previous findings of genetic influences on PPI, and provide novel insights into the molecular mechanisms regulating sensorimotor gating.