To evaluate the effects of pH control strategy on cell growth and the production of antibiotic (cyclo(2-Me-BABA-Gly)) byXenorhabdus nematophilaand enhance the antibiotic activity. The effects of uncontrolled- (different initial pH) and controlled-pH (different constant pH and pH-shift) operations on cell growth and antibiotic activity ofX. nematophilaYL00I were examined. Experiments showed that the optimal initial pH for cell growth and antibiotic production ofX. nematophilaYL001 occurred at 7.0. Under different constant pH, a pH level of 7.5 was found to be optimal for biomass and antibiotic activity at 23.71 g/L and 100.0 U/mL, respectively. Based on the kinetic information relating to the different constant pH effects on the fermentation ofX. nematophilaYL001, a two-stage pH control strategy in which pH 6.5 was maintained for the first 24 h, and then switched to 7.5 after 24 h, was established to improve biomass production and antibiotic activity. By applying this pH-shift strategy, the maximal antibiotic activity and productivity were significantly improved and reaching 185.0 U/mL and 4.41 U/mL/h, respectively, compared to values obtained from constant pH operation (100.0 U/mL and 1.39 U/mL/h).