We studied the regulation of the human Galβ1, 3GalNAc/Galβ1,4GlcNAc α2,3-sialyltransferase (hST3Gal IV) gene during HL-60 cell differentiation induced by dimethyl sulfoxide (DMSO), all trans-retinoic acid (ATRA), and phorbol myristate acetate (PMA). During differentiation, levels of hST3Gal IV mRNA dramatically decreased after 1 day of stimulation. Reverse-transcription PCR identified two mRNA isoforms, types B1 and BX, in HL-60 cells. The results of luciferase assays showed that the level of B3 promoter activity is high, whereas A1/2 and B2 promoter activities are low in HL-60 cells, suggesting that type B1, BX, and B3 mRNA isoforms are expressed in HL-60 cells. A luciferase assay identified a functional DNA portion within the proximal region of the B3 promoter that confers negative transcriptional regulation on the hST3Gal IV B3 promoter during HL-60 differentiation. These results suggest that this element plays a critical role in down-regulating the B3 promoter activity during HL-60 cell differentiation.