An important aspect of the cellular differentiation in the intestine is the migration of epithelial cells from the crypt to the villus tip. As homeodomaine transcription factor CDX2 has been suggested to influence cell migration, we performed a genome-wide promoter analysis for CDX2 binding in the differentiated human intestinal cancer cell line Caco-2 in order to identify CDX2-regulated genes involved in cellular migration. The engulfment and cell motility 3 (ELMO3) gene was identified as a potential CDX2 target gene. ELMO3 is an essential upstream regulator of the GTP-binding protein RAC during cell migration. However, no information is available about the transcriptional regulation of the ELMO3 gene. The aim of this study was to investigate the potential role of CDX2 in the regulation of the ELMO3 promoter activity. Electrophoretic mobility shift assays showed that CDX2 bound to conserved CDX2 sequences and mutations of the CDX2-binding sites, significantly reduced the promoter activity. Reporter gene assays demonstrated that the region mediating ELMO3 basal transcriptional activity to be located between -270 and -31 bp. Sequence analysis revealed no typical TATA-box, but four GC-rich sequences. In vitro analyses (electrophoretic mobility shift assays and promoter analyses) demonstrate that the SP1-binding sites are likely to play an important role in regulating the ELMO3 promoter activity. Furthermore, we showed here that CDX2 and SP1 can activate the ELMO3 promoter. Taken together, the present study reports the first characterization of the ELMO3 promoter and suggests a significant role of CDX2 in the basal transcriptional regulation of the intestine-specific expression of ELMO3, possibly through interaction with SP1.