To study the molecular basis of intramuscular fat (IMF) deposition, suppression subtractive hybridization was used to investigate the differences in gene expression between m. longissimus dorsi (LD) of high IMF Laiwu pig group and low IMF Laiwu pig group. From two specific subtractive cDNA libraries, the expression-upregulated clone HL-27 was selected by reverse Northern high-density blot, and then identified to be pig mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4). Pig NDUFS4 full-length cDNA was cloned by RACE, and contains a 528 bp-open reading frame (ORF) encoding 175 amino acid residues. The derived amino acid sequence of NDUFS4 is well conserved compared with NDUFS4 of various species with higher degree of sequence similarity with other mammalian (86.3-92.6 %) than amphibian, aves, and fishes (70.2-81.1 %), and contains one N-linked glycosylation site, one O-linked glycosylation site, seven Ser phosphorylation sites and five Thr phosphorylation sites. A-G mutation was found at nt 122 site of ORF between Laiwu pig and Large White, which results in the K-R mutation at 41 site of protein sequence. Real-time PCR analysis indicated that the level of NDUFS4 mRNA expression was higher in high IMF Laiwu pig group than in low IMF Laiwu pig group, and in Laiwu pig than in Large White. The tissue expression of the pig NDUFS4 gene showed a tissue-specific pattern: highly expressed in LD muscle, spleen and kidney, but hardly expressed in lung, stomach and large intestine. The possible role of NDUFS4 and its relation to IMF deposition are discussed.