The N-acetyltransferase 9 (NAT9) is an important reproduction related gene. In this study, we cloned the full-length cDNA sequence of porcine NAT9 gene through the rapid amplification of cDNA ends method. The porcine NAT9 gene encodes a protein of 206 amino acids which shares high homology with the NAT9 of seven species: rhesus monkey (87%), chimpanzee (87%), human (87%), horse (86%), rat (80%), mouse (79%) and gray short-tailed opossum (79%). This gene is structured in six exons and five introns as revealed by computer-assisted analysis. Tissue transcription profile analysis indicated that the porcine NAT9 gene is generally but differentially expressed in the detected tissues including spleen, large intestine, lung, fat, muscle, heart, liver, kidney and ovary. PCR-Alu I-RFLP was established to detect an A/G mutation on the position of 699-bp of mRNA and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus is significantly associated with the litter size of first parity (P < 0.05). This suggests that the NAT9 gene can be an useful candidate gene in selection for increasing litter size in pigs.