This study focuses on sirtuins, which catalyze the reaction of NAD+-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5 mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5 mM hydroxyurea and 50 µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.