Gamma-aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response toPseudomonas syringaeinArabidopsis thaliana. While searching for the mechanism underlying the pathogen-responsive mitogen-activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genesGAD1andGAD4. Inoculation withPseudomonas syringaepvtomatoDC3000 (Pst) andPst-avrRpt2expressing theavrRpt2effector gene inducedGAD1andGAD4gene expression and increased the levels of GABA. Genetic evidence revealed thatGAD1, GAD2, andGAD4play important roles in both GABA biosynthesis and plant resistance in response toPst-avrRpt2infection. Thegad1/2/4triple andgad1/2/4/5quadruple mutants, in which the GABA levels were extremely low, were more susceptible to bothPstandPst-avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction ofGAD1andGAD4expression afterPst-avrRpt2treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.