Monascus spp. are filamentous fungi, which can produce Monascus azaphilone pigments, monaklin K and other beneficial secondary metabolites, and they can also produce a mycotoxin citrinin. In this study, the gene Mrrpd3, encoding Rpd3-like histone deacetylase was overexpressed to evaluate its regulation on the production of pigments and mycotoxin in Monascus ruber. RT-qPCR confirmed the expression of Mrrpd3 was increased in the Mrrpd3-overexpressing strain. UPLC detection showed Mrrpd3-overexpressing strain enhanced the citrinin content by 61.9%, 56.5% and 52.6% at the 3rd day, 9th day and 11th day, respectively. But overexpression of Mrrpd3 had no significant effect on pigments production. RT-qPCR results showed that overexpression of Mrrpd3 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6 and mrl7. Western blot results suggested that overexpression of Mrrpd3 caused the decrease of acetylation modification of total protein and histone H3K27. Overall, these results indicate that Mrrpd3 plays an important role in regulating citrinin production of Monascus ruber. This study provides important insights into the mechanisms underlying the effects of the Mrrpd3 gene on the citrinin of Monascus ruber.