This paper describes the production of divalent K88, K99 antigens by high cell density fermentation and gene overexpression. The cell density reached above 40 at A600nm and the antigens were at 2(12) level. The thousands dosage of the vaccine can be made by using 10 I broth of the fermentation. The stability of the plasmid showed that about 30 percent of the bacteria lost its plasmid after 20 h fermentation. It was found that the antigens were overexpressed and located in both the pili of E. coli and in the medium in equal quantities. It means that the expression and regulation of the genes of K88, K99 may be different from the wild type of enterotoxingenic E. coli. A large number of the vaccinated pregnant sow showed that the piglets were effectively protected from the infection of enterotoxingenic E. coli. The results indicated that the large quantities requirement of the vaccine could be provided by using a small fermenter. This vaccine consists of two forms of the antigen K88, K99 which, when present in the pili as well as the medium, is more favorable to stimulate the production of antibody in the colostrum of pregnant sow.