Objectives Developing a counterselective system for efficient markerless gene deletions in biocontrol strain P. protegens Pf-5. Results We successfully implemented a markerless deletion of upp in Pf-5 to obtain the 5-FU resistant strain Pf5139. With this strain, we performed markerless gene deletions for each component of Gac/Rsm system and a 17 kb DNA fragment with the deletion ratio of 20 to 50%, and efficiently constructed a strain with triple deletions based on the suicide plasmid pJQ200UPP. In addition, there is no obvious connection between the deleted fragment length and the deletion ratio. Conclusion The upp-based counterselective system in this study is efficient and valuable for markerless gene deletions in Pf-5, indicating that it has great potential in the study of gene function and in the application of genome reduction for Pseudomonas strains.