Mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH3) catalyzes the allosterically regulated rate-limiting step of the tricarboxylic acid cycle activated. In pigs, very little is known about this gene. Here, we cloned 1,346 bp full-length cDNA and 8,778 bp genomic sequence of porcine gamma subunit of IDH3 (IDH3 gamma). IDH3 gamma contains 12 exons separated by 11 introns. Real-time PCR revealed that IDH3 gamma mRNA were upregulated in backfat of Large White compared with Meishan and F1 hybrids, and most abundant in small intestine via tissue distribution profile. A microsatellite ("GT" repeats) in second intron was found. The selected pigs were genotyped at this microsatellite. The IDH3 gamma genotypes showed a significant effect on backfat thickness at thorax-waist (P < 0.05), backfat thickness at sixth to seventh thorax (P < 0.01), and average backfat thickness (P < 0.05). This site seemed to be significantly dominant in action (P < 0.05 for backfat thickness at sixth to seventh thorax, backfat thickness at thorax-waist, and average backfat thickness), and allele B was associated with increase of thickness values of these traits. This locus is possibly considered as a marker for adipose deposition traits.