Malonyl-CoA is utilised in fatty acid biosynthesis by the enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT).

MCAT serves to transfer malonate from malonyl-CoA to the terminal thiol of holo-acyl carrier protein (ACP).

Malonyl-CoA is formed in the first step of mitochondrial fatty acid synthesis (mtFASII) from malonic acid by malonyl-CoA synthetase (ACSF3).

The enzyme MCAT together with an acyl carrier protein (ACP), and a polyketide synthase (PKS) and chain-length factor heterodimer, constitutes the minimal PKS of type II polyketides.

Malonyl-CoA is a highly regulated molecule in fatty acid synthesis; as such, it inhibits the rate-limiting step in beta-oxidation of fatty acids.

Malonyl-CoA inhibits fatty acids from associating with carnitine by regulating the enzyme carnitine acyltransferase, thereby preventing them from entering the mitochondria, where fatty acid oxidation and degradation occur.

Malonyl-CoA plays a special role in the mitochondrial clearance of toxic malonic acid in the metabolic disorder combined malonic and methylmalonic aciduria (CMAMMA).