Potted Italian ryegrasses (Lolium multiflorum L.) were used to investigate the effect of ammonia-oxidizing bacterial (AOB) strain that coexisted in rhizosphere soil on Italian ryegrass regrowth. The results showed that the isolated and screened AOB strain (S2_8_1) had 100% similarity to Ensifer sesbaniae. The inoculation of S2_8_1 on day 44 before defoliation caused its copy number in rhizosphere soils to increase by 83–157% from day 34 before defoliation to day 14 after defoliation compared with that in Italian ryegrass without S2_8_1 inoculation, indicating that S2_8_1 coexisted permanently with Italian ryegrass. The coexistence promoted the delivery of root-derived cytokinin to leaves and to increase its cytokinin concentrations; thus, the Italian ryegrass regrowth accelerated. During the 14-day regrowth period, the S2_8_1 coexistence with Italian ryegrass caused its leaf and xylem sap cytokinin concentrations, rhizosphere soil nitrification rates, net photosynthetic rates, and total biomass to increase by 38%, 58%, 105%, 18%, and 39% on day 14 after defoliation, respectively. The inoculation of S2_8_1 on day 2 before defoliation also increased the regrowth of Italian ryegrass. Thus, the coexistence of AOB with Italian ryegrass increased its regrowth by regulating the delivery of cytokinins from roots to leaves.