The ptsHIcrr operon of phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Escherichia coli DH5α was deleted to generate DH5α△ptsHIcrr strain, and its characteristics of growth and metabolism were compared with that of DH5α△ptsG. DH5α△ptsHIcrr and DH5α△ptsG were successfully constructed by using a one-step markerless deletion method that is a modified Red homologous recombination combined with specific cutting of I-SceⅠ. In LB medium, DH5α△ptsHIcrr has a significant different characteristic of growth with DH5α and DH5α△ptsG, and the maximum biomass of DH5α△ptsHIcrr is about two times of that of DH5α or DH5α△ptsG. However, there was no difference between the growth of DH5α and that of DH5α△ptsG. In LB medium supplemented with 1% glucose, DH5α△ptsHIcrr and DH5α△ptsG both grew better than DH5α, and the maximal cell densities were approximately 2 times and 2.8 times of that of DH5α respectively. In the end of culture, the concentrations of acetic acid of DH5α△ptsHIcrr, DH5α△ptsG were12.2%, 47% of that of DH5α. In a modified M9 medium, the specific growth rate (1/h) and the specific glucose consumption rate [g/(g·h)] of DH5α△ptsHIcrr were much slower than that of DH5α and a little slower than that of DH5α△ptsG. Altogether, the ptsHIcrr operon-deleted mutant changed the specific glucose consumption rate and showed many different characteristics of growth and metabolism from that of the △ptsG-deleted mutant.