Aflatoxin (AFT) contamination caused by Aspergillus flavus, a saprophytic aerobic fungus, in several crops such as peanut, maize and cottonseed is considered as the most serious factor influencing food safety concerning animal and human health worldwide. Whole genome sequences of A. flavus have been released and the genome size is about 37 Mb on 8 chromosomes encoding over 13 000 functional genes. In the whole genome sequences of A. flavus (NRRL3357), 55 putative secondary metabolite clusters have been identified based on backbone enzyme gene analysis by SMURF, and only three secondry metabolite clusters including AFT, cyclopiazonic acid (CPA) and aflatrem have been characterized. The above three secondary metabolite clusters are regulated by different environmental factors, secondary metabolite regulators, enzymatic activity, oxylipin and quorum sensing. AFT, CPA and aflatrem biosynthesis are inhibited by the global regulators, LaeA and VeA, in secondary metabolism. A family of oxylipin-producing oxygenases and their products encoded by ppo and lox can regulate sclerotia and conidia production and secondary metabolism in A. flavus. The profile of secondary metabolites could be influenced by variation of fungi density because higher fungi density will induce higher sporulation with decreased AFT. Most strains of A. flavus can produce numerous hydrolytic enzymes including α-amylases, pectinases, proteases, and lipases, which are believed to be important for fungal infection and virulence to host tissue. Further research on regulation of secondary metabolism and mycotoxins produced in A. flavus are also discussed.