Pseudomonas fluorescens 2P24 produces 2,4-diacetylphloroglucinol (2,4-DAPG) as the major antibiotic compound that protects plants from soil-borne diseases. Expression of the 2,4-DAPG biosynthesis enzymes, which are encoded by the phlACBD locus, is under the control of a delicate regulatory network. In this study, we identified a novel role for the outer membrane protein gene oprF, in negatively regulating the 2,4-DAPG production by using random mini-Tn5 mutagentsis. A sigma factor gene sigX was located immediately upstream of the oprF gene and shown to be a positive regulator for oprF transcription and 2,4-DAPG production. Genetic analysis of an oprF and sigX double-mutant indicated that the 2,4-DAPG regulation by oprF was dependent on SigX. The sigX gene did not affect PhlA and PhlD expression, but positively regulated the level of malonyl-CoA, the substrate of 2,4-DAPG synthesis, by influencing the expression of acetyl-CoA carboxylases. Further investigations revealed that sigX transcription was induced under conditions of salt starvation or glycine addition. All these findings indicate that SigX is a novel regulator of substrate supplements for 2,4-DAPG production.