Abstract We report here the identification and characterization of mouse GTPBP3 encoding a mitochondrial GTPase. A full-length GTPBP3 cDNA has been isolated and the genomic organization of GTPBP3 has been elucidated. The mouse GTPBP3 gene containing 9 exons encodes a 486 residue protein with a strong homology to the GTPBP3-like proteins of bacteria, yeast, and other homologs, related to tRNA modification. The mouse GTPBP3 is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, and brain. Surprisingly, this gene, unlike its human homolog, exhibited a low expression in skeletal muscle. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing GTPBP3–GFP fusion protein demonstrated that the mouse Gtpbp3 localizes in mitochondrion. These observations suggest that the mouse Gtpbp3 is an evolutionarily conserved mitochondrial GTP-binding protein involved in the tRNA modification. Thus, it may modulate the translational efficiency and accuracy of codon–anticodon base pairings on the decoding region of mitochondrial ribosomes.