LTRs of endogenous retroviruses are known to affect expression of several human genes, typically as a relatively minor alternative promoter. Here, we report that an endogenous retrovirus LTR acts as one of at least two alternative promoters for the human β1,3-galactosyltransferase 5 gene, involved in type 1 Lewis antigen synthesis, and show that the LTR promoter is most active in the gastrointestinal tract and mammary gland. Indeed, the LTR is the dominant promoter in the colon, indicating that this ancient retroviral element has a major impact on gene expression. Using colorectal cancer cell lines and electrophoretic mobility-shift assays, we found that hepatocyte nuclear factor 1 (HNF-1) binds a site within the retroviral promoter and that expression of HNF-1 and interaction with its binding site correlated with promoter activation. We conclude that HNF-1 is at least partially responsible for the tissue-specific activation of the LTR promoter of human β1,3-galactosyltransferase 5. We demonstrate that this tissue-specific transcription factor is implicated in the activation of an LTR gene promoter.