Histone acetyltransferase (HAT) has been reported to be pivotal for various physiological processes in many fungi. However, the functions that HAT Rtt109 perform in edible fungi Monascus and the underlying mechanism remains unclear. Here, we identified the rtt109 gene in Monascus, constructed the rtt109 knockout strain (Δrtt109) and its complementary strain (Δrtt109:com) by CRISPR/Cas9 methods, and functionally characterized the roles that Rtt109 play in Monascus. Deletion of rtt109 significantly reduced conidia formation and colony growth, whereas, it increased the yield of Monascus pigments (MPs) and citrinin (CTN). Further real-time quantitative PCR (RT-qPCR) analysis indicated that Rtt109 remarkably affected the transcriptional expression of key genes related to development, morphogenesis, and secondary metabolism of Monascus. Together, our results revealed the critical roles of HAT Rtt109 in Monascus, and enriched our current knowledge of the development and regulation of secondary metabolism in fungi, throwing light on restraining or eliminating citrinin in the development and industrial applications of Monascus.