Lewis Y (LeY) is a carbohydrate tumor-associated antigen. The majority of cancer cells derived from epithelial tissues express LeY type difucosylated oligosaccharides. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharides. In a previous study we reported that FUT4 is associated with cell proliferation; however, despite the important role of FUT4 in cancer proliferation and apoptosis, little is known about the mechanisms underlying the regulation of FUT4 transcription. In the current study we investigated the regulation of FUT4 transcription in human breast cancer. We compared the regulation of human FUT4 gene transcription in human breast cancer cells (MCF-7 and MDA-MB-231) using promoter/luciferase analyses. Using a series of promoter deletion constructs, we identified a potential regulatory site located between 0.8 and 1.6 kb of the FUT4 promoter. As shown by EMSA and ChIP analyses, heat-shock factor 1 (HSF1) and Sp1are required for FUT4 promoter activity. In addition, we explored the role of HSF1 and Sp1 on cell proliferation, and found that the ERK1/2 MAPK and PI3K/Akt signaling pathways regulate the expression of FUT4, which play a role in cell proliferation via HSF1 and Sp1. These results suggest that FUT4 is a target gene for HSF1 and Sp1 that is required for cell cycle progression in breast cancer epithelial cells. J. Cell. Biochem. 115: 168–178, 2014. © 2013 Wiley Periodicals, Inc.