In this study, we identified L-erythro-β-hydroxyasparagine (L-β-EHAsn) found abundantly in human urine, as a novel substrate of Zn2+-dependent D-serine dehydratase. L-β-EHAsn is an atypical amino acid present in large amounts in urine but rarely detected in serum or most organs/tissues examined. Quantitative analyses of urinary L-β-EHAsn in young healthy volunteers revealed significant correlation between urinary L-β-EHAsn concentration and creatinine level. Further, for in-depth analyses of L-β-EHAsn, we developed a simple three-step synthetic method using trans-epoxysuccinic acid as the starting substance. In addition, our research revealed a strong inhibitory effect of l-b-EHAsn on mammalian serine racemase, responsible for producing D-serine, a co-agonist of the N-methyl-D-aspartate receptor involved in glutamatergic neurotransmission.