The accumulation of eosinophils in inflammatory foci is a hallmark characteristic of Th2 inflammation. Nevertheless, the expression of inhibitory receptors such as paired immunoglobulin-like receptor B (PIR-B) and their function regulating eosinophil accumulation have received limited attention. We now report that Pirb was up-regulated in an eosinophil-dependent manner in the lungs of allergen-challenged and interleukin (IL)-13-overexpressing mice. Eosinophils expressed high levels of PIR-B, and Pirb(-/-) mice displayed increased gastrointestinal eosinophils. Consistent with these findings, PIR-B negatively regulated eotaxin-dependent eosinophil chemotaxis in vivo and in vitro. Surprisingly, Pirb(-/-) eosinophils and neutrophils had decreased leukotriene B4 (LTB(4))-dependent chemotactic responses in vitro. Furthermore, eosinophil accumulation was decreased in a chitin-induced model, partially dependent on LTB(4). Mechanistic analysis using a miniphosphoproteomic approach revealed that PIR-B recruits activating kinases after LTB(4) but not eotaxin stimulation. Consequently, eotaxin-activated Pirb(-/-) eosinophils displayed markedly increased extracellular signal-related kinase 1 and 2 (ERK1/2) phosphorylation, whereas LTB(4)-activated eosinophils had reduced ERK1/2 phosphorylation. We provide multiple lines of evidence supporting a model in which PIR-B displays opposing but potent regulatory functions in granulocyte activation. These data change the conventional wisdom that inhibitory receptors are restricted to inhibitory signals; we therefore propose that a single receptor can have dual functionality in distinct cell types after unique cellular signals.