Serine racemase (SR) is an enzyme that catalyzes the conversion of L-serine to pyruvate or D-serine, an endogenous agonist for N-methyl-D-aspartate (NMDA) receptors. Our previous study showed the presence of SR protein in the epidermis of wild type (WT) mice but not in SR knockout (KO) mice. Moreover, SR immune-reactivity was only found in the granular and cornified layers of epidermis in WT mice. It was suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of skin barrier. However, its role in skin barrier dysfunction like atopic dermatitis (AD) remains to be elusive. Therefore, this study was aimed to determine the content of D-serine in stratum corneum in AD patients and healthy controls, using tape stripping method. Skin samples were collected from cheek and upper arm of AD patient’s lesion and healthy persons. Significant difference was found in D-body ratio between AD involved, uninvolved and healthy control in cheek (p<0.005, p<0.001) and upper-arms (p<0.001, p<0.001), respectively. Furthermore, immune-histochemical analysis also revealed increased SR expression in the epidermis of AD patients compared to healthy control. In addition, keratinocytes stimulation with TNF-α or MIF, results in the increased expression of SR. Taken together, these findings suggest that D-serine particularly express in AD lesional skin and SR expression in the keratinocytes is linked to inflammatory cytokines.