Bacterial culture filtrates of an aggressive rhizobacterium, Pseudomonas chlororaphis O6, displayed strong nematicidal activity. The nematicidal activity of P. chlororaphis O6 was markedly reduced in the gacS mutant of P. chlororaphis O6 grown in the presence of glycine, but no reduction of nematicidal activity in the gacS mutant was noted in the absence of glycine. The results of bioassay with P. chlororaphis O6 mutants showed that phenazine and pyrrolnitrin production was not a major factor, but the effects of glycine in the culture medium suggest that formation of hydrogen cyanide might be important. Assessments in greenhouse studies with tomatoes growing in nematode-infested soils confirmed that the application of P. chlororaphis O6 resulted in the control of the root-knot nematode. Our results demonstrated that P. chlororaphis O6 could be employed as a biocontrol agent for the control of the root-knot nematode, and the global regulator, GacS, functions as a positive regulator of the expression of nematicidal compounds and enzymes in P. chlororaphis O6.