N-glycolylneuraminic acid (Neu5Gc), a generic form of sialic acid, is enzymatically synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Although expression of pig CMAH gene pcmah encoding CMAH has been reported to be regulated by pathogenic infection and developmental processes, little is known about the mechanisms underlying the regulation of pcmah gene expression. The objective of this study was to determine mechanism(s) involved in intestine specific regulation of pcmah gene by identifying several cis-acting elements and nuclear transcription factors that could directly interact with these cis-acting elements. We identified intestine specific promoter region (Pi) of pcmah gene located at upstream regions of the 5'flanking region of exon 1a and found that the promoter region is responsible for the transcriptional regulation of 5'pcmah-1. Based on reporter assays using serially constructed luciferase genes with each deleted promoter, we demonstrated that the Pi promoter activity was more active in intestinal IPI-2I cells than that in kidney PK15 cells, corresponding to both mRNA expression patterns in the two cell lines. In addition, we found that Sp1 transcription factor was necessary for basal activity of Pi promoter and that Ets-1 contributed to intestine-specific activity of Pi promoter. This study helps us understand transcriptional regulation of pcmah in the intestine of pig tissues. It also allows us to consider potential roles of Neu5Gc in interaction with environmental factors present in the intestinal tissue during pathogenic infection and developmental process.