The leukocyte-specific β2-integrin receptor family exerts a wide range of functions: β2- integrins are involved in leukocyte trafficking, where they mediate cell adhesion during inflammatory responses via binding to ICAM-1, ICAM-2, or JAM-C. Furthermore, they are essential for the recognition and phagocytosis of pathogens opsonized by complement. Accordingly, the β2-integrin family is known to be involved in autoimmune and inflammatory diseases, such as systemic lupus erythematosus. Owing to their complex biology, involving multiple conformational transitions, different signaling pathways, and a broad spectrum of ligands, the development of β2-integrin-targeted probes and therapeutics has remained challenging. We aimed to develop macrocyclic peptides, derived from phage display screening, which can be used to unravel ligand binding profiles of β2-integrins with an emphasis on the αI-domain. The selection of suitable lead peptides, and the characterization of their interaction profiles with different αI-domains, was enabled by an established in-vitro assay platform. Various peptide sequences were enriched during several rounds of phage display against the αI-domain of CR3, of which two peptides with particularly low micromolar binding affinity were further characterized. Both peptides showed direct binding to β2-integrin αI-domains and, in a competitive assay, dose-dependent inhibition of the αI-domains interactions with their main ligands iC3b and ICAM-1, respectively. These ligand-interfering properties were confirmed in bead- and cell-based adhesion assays. The modulators developed here are expected to provide valuable insight into the (patho-)physiology of CR3 and the other members of the β2-integrin family, as the two peptides were able to compete with different ligands. In the future, this may help to identify potential therapeutic approaches for autoimmune, inflammatory, and age-related diseases.