Syncytin, a protein encoded by an envelope gene of a human endogenous retrovirus-W (HERV-W), plays a critical role in trophoblast differentiation. We isolated the 5′-flanking region of the syncytin gene from human genomic DNA by PCR and identified cis-acting elements on the promoter that are important for transcription. The major transcription initiation site identified by mung bean nuclease protection assays is 56 base pairs (bp) downstream from a putative CCAAT box. Deletion analysis of the 5′-flanking region of the syncytin gene indicated that the proximal 148 bp are essential for minimal promoter activity and that regions of the promoter from nt −1519 to −984 and nt −294 to −148 are required for maximal expression in normal trophoblast cells. DNase I footprint analysis of the region between nt −252 and 110 revealed three protected regions, FP1–FP3. Mutagenesis of a hepatocyte-specific nuclear protein-1 (HAPF1) binding site in FP1 and a TATA box in FP3 had no effects on basal promoter activity. However, mutation of the CCAAT motif and the octamer protein (Oct) binding site in FP2 decreased promoter activity by 88% and 76%, respectively. Mutation of the ecdysone receptor (EcR) response element in FP2, which may bind a nuclear hormone receptor, increased basal promoter activity by 2-fold. Gel shift and supershift assays indicated that CCAAT-binding factor (CBF) binds to the CCAAT motif and that Oct binds to the Oct binding site. Taken together, these findings indicate that the syncytin promoter is located in the 5′ long terminal repeat (LTR) of the HERV-W gene and that binding sites for CBF and Oct in the proximal promoter are critical for transcriptional regulation of the gene in trophoblast cells.