The intramuscular fat content (IMF) is an economically important trait in pigs and the Laiwu pig is famous for its excessively extremely high level of IMF. Our previous transcriptome study revealed that the dynamic expression of glycerol-phosphate acyltransferase 3 (GPAT3) is consistent with changes in the IMF of Laiwu pigs. In this study, we further analyzed the expression and polymorphism of GPAT3 in its promoter region. The results indicated that the expression of GPAT3 increased dramatically from 120 to 240 days and is consistent with changes in IMF deposition, and at both mRNA and protein levels, GPAT3 expression was markedly higher in the LD muscle of Laiwu pigs than that of Duroc × Landrace × Yorkshire pigs. Deletion from −1695 to −1187 of porcine GPAT3 greatly increased its transcription. Of the two SNPs identified, the transition from C to T at −1526 site increased the transcription of porcine GPAT3 and allele T mainly distributed in Laiwu pig population. These results collectively suggest that the SNP at −1526 site of GPAT3 may contribute to IMF deposition by affecting its expression in pigs. [ABSTRACT FROM AUTHOR]