d-Aspartate is found in the nervous and reproductive system and participates in various physiological roles. While several lines of evidence suggest that this amino acid has an endogenous origin, the enzyme responsible for mammalian d-Asp biosynthesis has not yet been identified. We show that mammalian serine racemase (SRR), the primary enzyme responsible for brain d-Ser production, catalyses Asp racemization via a two-base mechanism. We observed that overexpression of SRR in rat pheochromocytoma PC12 cells resulted in an increase in intracellular d-Asp compared with control cells, demonstrating that SRR functions as an Asp racemase in the cells. To investigate the impact of endogenous SRR on endogenous d-Asp levels in the cells, we generated SRR-knockout (SRR-KO) PC12 cells. The SRR-KO cells exhibited decreased intracellular d-Ser levels, but production levels of d-Asp were unaffected. In contrast, SRR-KO mice showed significantly decreased d-Asp levels in their frontal cortices and hippocampi, where SRR is normally highly expressed, while d-Asp levels in the cerebellum and testes remained unchanged. Our results indicate that SRR indeed acts as a d-Asp biosynthetic enzyme in some organs and/or tissues, and also provide evidences that there should be some additional enzyme for d-Asp synthesis in mammals.