[Objective] DNA phosphorothioate (PT) modification, in which the sulfur replaces a nonbridging oxygen, occurs naturally in diverse bacteria, archaea and human pathogens as a new kind of epigenetic modification. However, the regulatory mechanism of PT modification has not been fully characterized. In this study, the regulatory mechanism of spfB on DNA phosphorothioate (PT) modification in Pseudomonas fluorescens Pf0-1 was demonstrated. [Methods] Firstly, the spfB genetic interruption and complementary strains were constructed by homologous recombination and then tested the modification frequency in these strains by iodine cleavage. The operons within spf gene cluster were grouped by RT-PCR and the transcriptional level was analyzed in the ΔspfB mutant by quantitative real-time PCR. Finally, the possible regulatory region of SpfB on the spf operon was characterized by EMSA and DNase I footprinting assay.[Results] The inactivation of spfB led to more dispersed small fragments in genomic DNA of ΔspfB mutant and its complementation obviously restored the phenotype of wild type strain. Genes in spf gene cluster were assigned into one co-transcription unit, and the disruption of spfB directly up-regulated the transcription of the operon. In vitro SpfB directly protected two separate sequences within the spf promoter region from DNase I cleavage, and each protected sequence contained a direct repeat (5'-TGTTTGT-3'). [Conclusion] SpfB in Pseudomonas fluorescens Pf0-1 was a negative regulator in DNA phosphorothioate modification.