Four αI domain-containing integrins (α1β1, α2β1, α10β1, and α11β1) have evolved to recognize various members of the collagen family. A defining structural feature of these receptors is the presence of the αC helix within their αI domains (Glu284–Arg288 in the α2I domain), a structure solely found in collagen-binding integrins, whose functional mechanism has remained unclear. To elucidate the functional role of the αC helix, and to assess the contribution of the mechanistically important Arg288-Glu318 ion pair in α2I domain activation, we created two variants α2IR288A and α2IE318A. Functional solid-phase binding assays and surface plasmon resonance revealed that these variants exhibit strikingly increased avidity for collagens I and IV, whereas affinities for triple-helical GFOGER peptides, representing a single binding motif, were only slightly increased. The variants displayed distinct ligand-binding profiles, including differences in association and dissociation constants. To understand these differences at a structural level, we determined four novel X-ray crystal structures of the variants, including both ligand-free and ligand-bound (succinic acid or malic acid) states. These structures revealed differences in the folding of the αC helix region, suggesting that its conformation regulates α2I domain activation and ligand specificity, consistent with our binding assays. Additionally, the structures captured the movements of the catalytic metal ion in the metal ion dependent adhesion site, providing a detailed view of the sophisticated activation mechanism of the α2I domain.