Abstract Background Generally, bacteria have a circular genome with a single replication origin for each replicon, whereas archaea and eukaryotes can have multiple replication origins in a single chromosome. In Escherichia coli, bidirectional DNA replication is initiated at the origin of replication (oriC) and arrested by the 10 termination sites (terA–J). Results We constructed E. coli derivatives with additional or ectopic replication origins, which demonstrate the relationship between DNA replication and cell physiology. The cultures of E. coli derivatives with multiple replication origins contained an increased fraction of replicating chromosomes and the cells varied in size. Without the original oriC, E. coli derivatives with double ectopic replication origins manifested impaired growth irrespective of growth conditions and enhanced cell size, and exhibited excessive and asynchronous replication initiation. The generation time of an E. coli strain with three replication origins decreased in a minimal medium supplemented with glucose as the sole carbon source. As well as cell growth, the introduction of additional replication origins promoted increased biomass production. Conclusions Balanced cell growth and physiological stability of E. coli under rapid growth condition are affected by changes in the position and number of replication origins. Additionally, we show that, for the first time to our knowledge, the introduction of replication initiation sites to the chromosome promotes cell growth and increases protein production.