Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαiβγ protein-dependent and β-arrestin-related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gβγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. β-arrestin2 recruitment was studied in OXE-R-overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks β-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE-triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE-induced Ca(2+) flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gβγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi-biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.