The appearance of plasmid-losing cells in a recombinant Escherichia coli culture was observed when the cell mass became doubled after induction, which corresponded to the timing of cell fission. Accordingly, a two-stage fermentation strategy capable of maintaining plasmid stability without selective pressure in a recombinant E. coli culture was proposed. In the first stage (cell growth stage), a high cell density culture was obtained by incubating the cells in the R medium. In the second stage (producing stage), the cells were devoted to producing the recombinant protein by introducing the fresh LB medium supplemented with isopropyl-β-D-thiogalactopyranoside (IPTG). It was necessary to prevent the doubling in the cell mass after induction; otherwise cell fission would occur and generate plasmid-losing cells. The present strategy is expected to be extensively applicable in recombinant E. coli cultures.