Polyunsaturated fatty acids (PUFAs) are essential for brain development and function. Increasing evidence has shown that an imbalance of PUFAs is associated with various human psychiatric disorders, including autism and schizophrenia. Fatty acid-binding proteins (FABPs), cellular chaperones of PUFAs, are involved in PUFA intracellular trafficking, signal transduction, and gene transcription. In this study, we show that FABP3 is strongly expressed in the GABAergic inhibitory interneurons of the male mouse anterior cingulate cortex (ACC), which is a component of the limbic cortex and is important for the coordination of cognitive and emotional behaviors. Interestingly,Fabp3KO male mice show an increase in the expression of the gene encoding the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (Gad67) in the ACC. In the ACC ofFabp3KO mice,Gad67promoter methylation and the binding of methyl-CpG binding protein 2 (MeCP2) and histone deacetylase 1 (HDAC1) to theGad67promoter are significantly decreased compared with those in WT mice. The abnormal cognitive and emotional behaviors ofFabp3KO mice are restored by methionine administration. Notably, methionine administration normalizesGad67promoter methylation and its mRNA expression in the ACC ofFabp3KO mice. These findings demonstrate that FABP3 is involved in the control of DNA methylation of theGad67promoter and activation of GABAergic neurons in the ACC, thus suggesting the importance of PUFA homeostasis in the ACC for cognitive and emotional behaviors.SIGNIFICANCE STATEMENTThe ACC is important for emotional and cognitive processing. However, the mechanisms underlying its involvement in the control of behavioral responses are largely unknown. We show the following new observations: (1) FABP3, a PUFA cellular chaperone, is exclusively expressed in GABAergic interneurons in the ACC; (2) an increase inGad67expression is detected in the ACC ofFabp3KO mice; (3) theGad67promoter is hypomethylated and the binding of transcriptional repressor complexes is decreased in the ACC ofFabp3KO mice; and (4) elevatedGad67expression and abnormal behaviors seen inFabp3KO mice are mostly recovered by methionine treatment. These suggest that FABP3 regulates GABA synthesis through transcriptional regulation ofGad67in the ACC.