The technology of gene knockout using lambda Red system in bacteria has been developed lately, but its application being limited to Esherichia coli. In order to get some experiences in other bacteria we select four genes, including alkA, wcaJ, yphF and dam, to test gene knockout using Red system in E. coli and Shigella. As a result, three genes were successfully knocked out in E. coli except dam while only alkA gene was knocked out in Shigella. It showed that the Red system should be improved if it was to be effectively used in Shigella and other bacteria.