Acetyl-CoA synthetase (AceCS) catalyzes the production of acetyl-CoA from acetate, CoA and ATP. There are two types of AceCS in mammals with different functions. One designated AceCS1 is a cytosolic enzyme expressed in the liver and plays a role in the production of acetyl-CoA for the synthesis of fatty acids and cholesterol. The other enzyme AceCS2 is a mitochondrial matrix enzyme that produces acetyl-CoAs mainly utilized for oxidation. Consistent with its function, the transcription of AceCS1 is regulated by SREBPs. In contrast, the expression of AceCS2 is upregulated during starvation and ketogenesis via unknown mechanisms. Specific inhibitors of AceCS may provide therapeutic agents for the treatment of obesity, cardiovascular diseases and type 2 diabetes.