The peroxisomal 3-oxoacyl-CoA thiolase (thiolase) is the enzyme involved in the last step of β- oxidation of fatty acids to acyl-CoA and acetyl-CoA. The enzyme is nuclear encoded, synthesized in the cytoplasm and imported into peroxisomes. The importance of studying thiolase is that it generates acetyl-CoA which is a precursor for the synthesis of molecules like cholesterol and other fatty acids. The structural and functional analysis of thiolase at the molecular level may add to the knowledge of fatty acid metabolism. It is known that several genes mediate lipid homeostasis in target organs like liver and adipose tissue and are regulated by peroxisome proliferator-activated receptor (PPAR). This review will focus on the structural and functional aspects of thiolase, its isoforms, and their comparison at the molecular level.