The human anterior cingulate cortex (ACC) is part of the default-mode network that shows predominant negative blood oxygen level-dependent (BOLD) responses in functional magnetic resonance imaging (fMRI). We combined fMRI during emotional processing and resting-state magnetic resonance spectroscopy measurements and observed that the concentration of GABA in the ACC specifically correlated with the amount of negative BOLD responses in the very same region. Our findings show that default-mode network negative BOLD responses during emotions are mediated by GABA.