Background: Glycosyltransferase inhibitors have important applications in therapeutics and as chemical biology tools.Results: The human β1,4-galactosyltransferase 7 enzyme active site was mapped by modeling, mutagenesis, and in vitro/in cellulo assays, and novel inhibitors were synthesized.Conclusion: An efficient inhibitor of β1,4-galactosyltransferase 7 and glycosaminoglycan synthesis was obtained.Significance: This inhibitory molecule can be exploited to investigate glycosaminoglycan biology and modulate glycosaminoglycan synthesis in therapeutics.