Technical Abstract: Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi, Aspergillus flavus and Aspergillus parasiticus. Aflatoxin biosynthesis is a quite complex process involving many intermediates and enzymes, regulated at multi-levels. Scientists from biochemistry, molecular biology, and genetics are attracted to the study of its mechanisms of biosynthesis and regulation. Significant progress has been made in the last decade. We have reviewed the aflatoxin biosynthetic pathway, pathway enzymes, and the genes encoding these enzymes involved in the conversion of major aflatoxin intermediates. Genes confirmed to be involved in regulation of aflatoxin formation and control of aflatoxin contamination are summarized as well.