Red yeasts demonstrate considerable potential in industrial and biotechnological applications, particularly in the biosynthesis of carotenoids and lipids, which are valuable secondary metabolites with a wide range of applications. In the oleaginous red yeast Rhodosporidium kratochvilovae YM25235, the acetyl-CoA synthetases RkACS1 and RkACS2 play critical roles in converting acetate into acetyl-CoA, a key precursor for the synthesis of various metabolites, including carotenoids and lipids. This study explores the physiological functions and metabolic regulation of RkACS1 and RkACS2, revealing distinct roles for these isoenzymes in metabolic processes. RkACS1 is essential for utilizing non-fermentable carbon sources such as acetate, ethanol, and glycerol, exhibiting high affinity for acetate and being activated by acetate while inhibited by glucose. Additionally, RkACS1 is involved in carotenoid biosynthesis. In contrast, RkACS2, while not specific to particular carbon sources, is primarily involved in lipid and fatty acid synthesis. It also influences gene expression through histone acetylation in the nucleus. Notably, these two isoenzymes exhibit functional redundancy and mutual regulation. These findings provide valuable insights into the metabolic regulation of acetyl-CoA synthesis, offering a foundation for engineering strategies aimed at optimizing secondary metabolite production in oleaginous red yeasts. KEY POINTS: • RkACS1 is related to carotenoid biosynthesis and essential for non-fermentable carbon sources • RkACS2 supports lipid and fatty acid biosynthesis and regulates histone acetylation in the nucleus • Functional redundancy and mutual regulation exist between RkACS1 and RkACS2 isoenzymes.