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 fungiMonascusand the underlying mechanism remains unclear. Here, we identified thertt109gene inMonascus, constructed thertt109knockout strain (Δrtt109) and its complementary strain (Δrtt109:com) by CRISPR/Cas9 methods, and functionally characterized the roles that Rtt109 play inMonascus. Deletion ofrtt109significantly reduced conidia formation and colony growth, whereas, it increased the yield ofMonascuspigments (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 ofMonascus. Together, our results revealed the critical roles of HAT Rtt109 inMonascus, 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 ofMonascus.