Background: The mechanism of action of olanzapine in treating schizophrenia is not clear. This research reports the effects of a therapeutic equivalent treatment of olanzapine on DNA methylation in a rat model in vivo. Genome-wide DNA methylation was assessed using a MeDIP-chip analysis. All methylated DNA immunoprecipitation (MeDIP), sample labelling, hybridization and processing were performed by Arraystar Inc (Rockville, MD, USA). The identified gene promoters showing significant alterations to DNA methylation were then subjected to Ingenuity Pathway Analysis (Ingenuity System Inc, CA, USA). Results: The results show that olanzapine causes an increase in methylation in 1,140, 1,294 and 1,313 genes and a decrease in methylation in 633, 565 and 532 genes in the hippocampus, cerebellum and liver, respectively. Most genes affected are tissue specific. Only 41 affected genes (approximately 3%) showed an increase and no gene showed a decrease in methylation in all three tissues. Further, the two brain regions shared 123 affected genes (approximately 10%). The affected genes are enriched in pathways affecting dopamine signalling, molecular transport, nervous system development and functions in the hippocampus; ephrin receptor signalling and synaptic long-term potentiation in the cerebellum; and tissue morphology, cellular assembly and organization in the liver. Also, the affected genes included those previously implicated in psychosis. Conclusions: The known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms as well as accounting for certain adverse effects including the metabolic syndrome. The results give insights into the mechanism of action of olanzapine, therapeutic effects and the side effects of antipsychotics. © 2014 Melka et al.; licensee BioMed Central Ltd.