N-Glycosylation of integrin alpha5beta1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (alpha5S3-5) on the beta-propeller of the alpha5 subunit are essential to the functional expression of the subunit. In particular, site 5 (alpha5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin beta1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the beta1 subunit (i.e. the Delta4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the beta1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either alpha5S3-5 or alpha5S5 mutant of the alpha5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the beta1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the alpha5S3-5/beta1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the beta1 subunit, it might also be useful for the study of molecular structures.