Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on β1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with α2-6 sialic acid and without α2-6 sialic acid, on the structure of the I-like domain of the b1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of β1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the α1 and 7 helices, which are important for the activation of β1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function. © 2008 Wiley-Liss, Inc.