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Summary The biosynthesis of specialized metabolites is strictly regulated by environmental inputs such as the day–night cycle, but the underlying mechanisms remain elusive. InPetunia hybridacv. Mitchell flowers, the biosynthesis and emission of volatile compounds display a diurnal pattern with a peak in the evening to attract nocturnal pollinators.Using petunia flowers as a model system, we found that chromatin level regulation, especially histone acetylation, plays an essential role in mediating the day–night oscillation of the biosynthetic gene network of specialized metabolites.By performing time‐course chromatin immunoprecipitation assays for histone modifications, we uncovered that a specific group of genes involved in the regulation, biosynthesis, and emission of floral volatile compounds, which displays the greatest magnitude in day–night oscillating gene expression, is associated with highly dynamic histone acetylation marks H3K9ac and H3K27ac. Specifically, the strongest oscillating genes featured a drastic removal of histone acetylation marks at night, potentially to shut down the biosynthesis of floral volatile compounds during the morning when they are not needed. Inhibiting daytime histone acetylation led to a compromised evening induction of these genes.Overall, our study suggested an active role of chromatin modification in the diurnal oscillation of specialized metabolic network.