Tobacco weather fleck is a kind of non-infective disease caused by O₃ injury at low temperature. Plant growth-promoting rhizobacteria (PGPRs) such as Bacillus velezensis confer benefits to crops by producing volatile organic compounds to trigger induced systemic tolerance (IST). Here, B. velezensis GJ11 was isolated and screened within the tobacco root, which can produce 2,3-butanediol and acetoin to trigger IST against O₃ injury in tobacco plants. Further analysis found acetoin played a more important role in triggering IST against O₃ injury than 2,3-butanediol. For a higher acetoin production, the bdh gene was deleted to block the metabolic pathway from acetoin to 2,3-butanediol in GJ11. The knockout strain GJ11∆bdh produced acetoin about four times more than GJ11, showing a much stronger activity against O₃ injury than the wild-type strain. Both acetoin and GJ11∆bdh could effectively enhance the antioxidant enzymes activity (e.g. SOD and CAT) in tobacco that was favourable for scavenging reactive oxygen species such as H₂O₂ in leaves after exposure to O₃. Thereby, less H₂O₂ was observed in the tobacco leaves pre-treated with acetoin or GJ11∆bdh. The result of field experiments also paralleled the result of the laboratory experiment. Both acetoin and GJ11∆bdh could well protect tobacco plants from O₃ injury in the high mountain areas after application by root-drench. The results presented in this study, from both the laboratory and fields, can broaden our knowledge on the application of PGPRs and their volatiles such as acetoin to protect crops from O₃ injury.