The proper development and function of telencephalic GABAergic interneurons is critical for maintaining the excitation and inhibition (E/I) balance in cortical circuits. Glutamate contributes to cortical interneuron (CIN) development viaN-methyl-d-aspartate receptors (NMDARs). NMDAR activation requires the binding of a co-agonist, either glycine ord-serine.d-serine (co-agonist at many mature forebrain synapses) is racemized by the neuronal enzyme serine racemase (SR) froml-serine. We utilized constitutive SR knockout (SR−/−) mice to investigate the effect ofd-serine availability on the development of CINs and inhibitory synapses in the prelimbic cortex (PrL). We found that most immature Lhx6 + CINs expressed SR and the obligatory NMDAR subunit NR1. At embryonic day 15, SR−/−mice had an accumulation of GABA and increased mitotic proliferation in the ganglionic eminence and fewerGad1+ (glutamic acid decarboxylase 67 kDa; GAD67) cells in the E18 neocortex. Lhx6 + cells develop into parvalbumin (PV+) and somatostatin (Sst+) CINs. In the PrL of postnatal day (PND) 16 SR−/−mice, there was a significant decrease in GAD67+ and PV+, but not SST + CIN density, which was associated with reduced inhibitory postsynaptic potentials in layer 2/3 pyramidal neurons. These results demonstrate that D-serine availability is essential for prenatal CIN development and postnatal cortical circuit maturation.