Oxysterols such as 7β-hydroxycholesterol (7β-HC) and 7-ketocholesterol (7-KC) have been linked to the pathophysiology of neurodegenerative diseases. This study was carried out to examine the effect of oxysterols on global gene expression in the rat prefrontal cortex (PFC). 7β-HC, 7-KC, or cholesterol was injected into the rat PFC and RNA was extracted from this brain region at 24-h post-injection and analyzed. Microarray analyses identified 1365 genes, whose expressions were affected by both 7β-HC and 7-KC. Among these, down-regulated genes outnumbered up-regulated genes. Pathway analysis showed that down-regulated genes had roles in carbohydrate metabolism, cell signaling and nucleic acid metabolism; and the majority of these encode G-protein coupled receptors (GPCRs). Expression of selected genes were validated by quantitative real-time PCR. Western blots confirmed down-regulation of oxytocin receptor (Oxtr) at 1 day post-7β-HC treatment. Immunohistochemical analysis showed localization of Oxtr in neurons of the PFC. Electron microscopy identified the presence of Oxtr-immunoreactivity in axon terminals. Together, these findings provide insights into molecular mechanisms through which oxysterols could exert their pathophysiological effects, and suggest that increased oxysterols may affect synaptic function by transcriptional repression of GPCRs.