Pseudomonas fluorescens 2P24, a biocontrol agent for soil-borne diseases has been isolated from the wheat take-all decline soil, and was characterized with efficient production of antifungal compounds. In this study, the gacS gene was cloned by PCR from the P. fluorescens 2P24 genomic library. Nucleotide sequencing indicated that the gacS gene contains 2754 bp, and is predicted to encode a peptide of 917 amino acids with molecular mass of 101 kD. The deduced amino acid sequence shares 91% identity to that of the sensor kinase GacS of P. chlororaphis and 89% identity to GacS of P. fluorescens CHAO. Further research revealed that the gacS-knock-out mutant was unable to produce anti-fungal metabolites, such as 2,4-DAPG, HCN and proteinase. Moreover, it lost the ability to inhibit the growth of Gaeumannomyces graminis in Petri dish and to control the development of wheat take-all disease in greenhouse. The complemented strain containing a plasmid-bearing gacS gene could restore all of the lost phenotypes. The results indicated that GacS in an important regulatory factor and directly controls the synthesis of key biocontrol factors and the biocontrol efficacy of P. fluorescens 2P24.