1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR, EC: 1.1.1.267) is the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and actually catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length DXR cDNA sequence (GenBank accession number: AY443101) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of GbDXR was 1720 bp containing a 1431 bp open reading frame (ORF) encoding a peptide of 477 amino acids with a calculated molecular mass of 52 kDa and an isoelectric point of 6.58. Comparative and bioinformatic analyses revealed that GbDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that GbDXR was more ancient than other plant DXRs. Tissue expression pattern analysis indicated that GbDXR expressed in all tissues including roots, stems, leaves, pericarps and seeds and lower transcription level was observed in leaves of G. biloba than that of other tissues. The cloning and characterization of GbDXR will be helpful to understand more about the role of DXR involved in the ginkgolides biosynthesis at the molecular level.