The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B protein on integrin-mediated signaling in primary murine myeloid cells. The pir-b−/− neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-α. The pir-b−/− and wild-type cells responded equivalently when stimulated with TNF-α in suspension, indicating that the hyperresponsive phenotype of the pir-b−/− cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-b−/− neutrophils expressed similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-b−/− mice were also hyperadhesive and spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular β 2 integrins. Biochemical analysis of macrophages from pir-b−/− mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling. These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling.