Glycerophosphodiester phosphodiesterase (GDPD) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. GDPD5 has been reported in Mus musculus and Gallus gallus, but not in Homo sapiens. Here we report the cloning and characterization of a novel human GDPD domain-containing gene, GDPD5, isolated from human testis cDNA library, and mapped to 11q13.4-13.5 by searching the UCSC genomic database. The GDPD5 cDNA sequence of 3442 base pairs contains an open reading frame encoding 605 amino acids. The GDPD5 gene consists of 17 exons and encodes a putative protein with six transmembrane regions and a GDPD motif. Subcellular localization of GDPD5 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7 cells. RT-PCR analysis showed that GDPD5 was widely expressed in human tissues and the expression levels in kidney and prostate were relatively low.