Peanut (Arachis hypogaea) allergy is one of the most serious food allergies. Peanut seed protein, Ara h 3, is considered to be one of the most important peanut allergens. Little is known about the temporal and spatial regulation mechanism of Ara h 3 during seed development. In this study, chromatin structure of the Ara h 3 promoter was analyzed to examine its transcriptional regulation. Analysis of transgenic plants of Arabidopsis thaliana expressing Arah3: GUS showed that the Ara h 3 promoter could efficiently direct the seed-specific expression of the GUS reporter gene. Chromatin immunoprecipitation revealed that nucleosomes were depleted at the core promoter of the Ara h 3 upon full activation in the late stage of embryo maturation, which was accompanied by a dramatic decrease of histone acetylation. However, in the early stage of embryo maturation, histone H3 hyperacetylation at the core promoter of Ara h 3 was detected. A decrease of histone H3-K9 dimethylation levels at core promoter of Ara h 3 was also observed with concomitant repression of Ara h 3 in the vegetative tissues. Our results suggest that the histone modification status of Ara h 3 undergoes targeted changes including the increase of histone H3 acetylation and decrease of histone H3-K9 dimethylation in early maturation embryos. In addition, the loss of histone H3 from the proximal promoter of Ara h 3 is associated with its high expression during late embryo maturation.