In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is crucial for the expression of secondary metabolism and the biological control of fungi, nematodes, and insects. Based on the findings of a previous metabolomic study, the role of intracellular γ-aminobutyrate (GABA) as a potential signal in the Gac/Rsm pathway was investigated herein. The function and regulation of a gabDT (c01870-c01880) gene cluster in strain CHA0 were described. The gabT gene encoded GABA transaminase (GABAT) and enabled the growth of the bacterium on GABA, whereas the upstream gabD gene (annotated as a gene encoding succinic semialdehyde dehydrogenase) had an unknown function. A gacA mutant exhibited low GABAT activity, leading to the markedly greater intracellular accumulation of GABA than in the wild type. In the gacA mutant, the RsmA and RsmE proteins caused translational gabD repression, with concomitant gabT repression. Due to very low GABAT activity, the gabT mutant accumulated GABA to high levels. This trait promoted a planktonic lifestyle, reduced biofilm formation, and favored root colonization without exhibiting the highly pleiotropic gacA phenotypes. These results suggest an important role of GABA in the Gac/Rsm-regulated niche adaptation of strain CHA0 to plant roots.