Endogenous D-serine is known to modulate glutamatergic transmission via interaction with the glycine site of N-methyl-D-aspartate (NMDA) receptors. D-serine is synthesized by racemization of L-serine using an enzymatic reaction catalyzed by serine racemase. Although much attention has been focused on the role of D-serine within the central nervous system, the physiological role of D-serine in enteric nervous system has not been investigated. Lower esophageal sphincter (LES) function is known to be modulated by NMDA-dependent mechanisms. The present study was aimed to study the expression of enzymes involved in D-serine metabolism and the function of D-serine in lower esophageal sphincter in rats. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting showed the expression of serine racemase in isolated rat LES. Electrical field stimulation was used to induce non-adrenergic non-cholinergic (NANC) contraction/relaxation of isolated rat LES in an organ bath using an isometric force transducer. The organ bath studies on isolated rat LES showed that incubation with D-serine (100 μM) is associated with a significant increase in the NANC contraction of isolated LES. This effect of exogenous D-serine was inhibited by NMDA receptor antagonists (MK-801), suggesting that NMDA receptors are involved in the effects of D-serine on NANC contraction of LES. Incubation with D-serine did not show a significant effect on NANC relaxation within our experimental setting. The results of this study suggest that serine racemase is expressed in LES and D-serine modulates contraction of the lower esophageal sphincter in rats.