Co-enzyme A is the essential cellular carrier of the acetyl group, largely used in fatty acid synthesis and lysine post-translational modifications of proteins. Following the analysis of the cellular CoA-binding proteome, we identified Nme1/2 as major abundant cytoplasmic CoA-binding proteins. We report a novel mechanism that controls fatty acids synthesis involving ATP-dependent acetyl-CoA binding by Nucleoside Diphosphate Kinases 1 and 2 (Nme1/2).