Acetyl-coenzyme A (acetyl-CoA) is a key metabolite for energy metabolism and biosynthetic pathways produced by acetyl-CoA synthetase (ACS) gene. In previous study, ACS gene were speculated to be related to metabolites production of Monascus sp. In this study, the expression level of ACS gene was inhibited by RNAi. 10 transformants were picked out to measure the expression level of ACS gene by RT-qPCR. The ACS gene expression level of transformant T7 was the lowest, 0.401 times higher than that of the wild strain. So T7 was selected for the follow-up study. The effects on the colonial morphology of Monascus had been observed. And the morphology of mycelia and spores were observed by scanning electron microscope. The contents of main secondary metabolites of Monascus- MK, citrinin were determined by HPLC. And the color values of wild-type strain and T7 transformant have been measured to reflect the content of pigments by spectrophotometer. The results showed that low expression level of ACS gene by RNAi could inhibit the growth of Monascus colonies and could affect the mycelial growth and the type of pigments. The low expression level of ACS gene could affect the formation and morphology of M. buliginosus M1 spores. It also could inhibit all the production of main secondary metabolites of Monascus-MK, citrinin and pigment at the same time.