Preoxisomal acyl-CoA oxidase 1 (ACOX1) catalyses the beta-oxidation of very long chain fatty acids and thus plays an essential role in fatty acid degradation. The objective of this study was to clone a gene encoding ACOX1 and to characterize its association with fat deposition in pigs. The complete coding sequence was 1986bp and encoded a protein of 661 amino acids including the carboxyl-terminal sequence (Ser-Lys-Leu) known as a minimal peroxisome-targeting signal. The predicted porcine ACOX1 amino acid sequence shared 88, 84, 81, 86 and 78% identity to that of human, mouse, rat, bovine and chicken counterparts, respectively. The A/C polymorphism in intron 9 was detected by PCR-PstI-RFLP and the allele frequencies of this polymorphism were significantly different between Chinese fatty type pig breeds (Meishan, Erhualian, Tongcheng and Qinping) and western commercial pig breeds (Landrace, Large White and Duroc). The association analysis between the A/C polymorphism and carcass traits in 298 Large White × Meishan F₂ offspring showed that the single genotype polymorphism (SNP) genotype was significantly associated with traits related to fat deposition such as backfat thickness and carcass fat percentage. The ACOX1 gene was widely expressed in the tissues examined by semi-quantitative reverse transcription (RT) PCR, with the highly abundant in liver, kidney and fat. The present study offered preliminary evidence to further determine the role of the ACOX1 gene in pig fat deposition.