<p>Subacute rumen acidosis (SARA) is a common metabolic disorder in dairy cows fed high-concentrate (HC) diets in intensive farming systems. No typical clinical sign of illness but some common signs include decreased dry matter intake, laminitis and diarrhea are observed in HC cows indicating a certain stressful and depressive behavior. However, transcriptional change in energy metabolic center of the hippocampus and hypothalamus in HC cows has not been reported. Twelve mid-lactation dairy cows were randomly assigned to a HC group (60% concentrate, <i>n</i> = 6) or a low-concentrate (LC) group (40% concentrate, <i>n</i> = 6) for 21 days. At the end of the feeding trial, hippocampus and hypothalamus tissues were collected from dairy cows fed a HC or LC diet for transcriptomic analysis. To verify transcriptomic findings, murine hypothalamic N38 cells were used to investigate the regulatory effect of thyroid hormone on gamma-aminobutyric acid (GABA) synthesis in vitro. In the hippocampus, RNA sequencing results revealed that differentially expressed genes (DEGs) were primarily enriched in neuroactive ligand-receptor interaction, serotonergic, and dopaminergic synapse pathways. In the hypothalamus, DEGs were significantly enriched in the neuroactive ligand-receptor interaction and GABAergic synapse pathways. In the hypothalamus of HC cows, the levels of GABA and its key synthetic enzyme GAD65 were significantly reduced, along with decreased free T3 (FT₃) concentration and THRB receptor protein expression. In vitro, T₃ significantly upregulated both mRNA and protein expression levels of GAD65 and THRB. Under THRB gene silencing, T₃ treatment failed to increase the mRNA and protein expression of THRB and GAD65 in hypothalamic N38 cells. These findings suggest that a HC diet alters neurotransmitter pathways and reduces T3 levels and THRB expression in the hypothalamus, which may impair GABA synthesis.</p>