The complement anaphylatoxins C3a, C5a, and desarginated C5a (C5a(desArg)) play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3a receptor and C5a receptor (C5aR). The role of C5a(desArg) in inducing cell activation has been often neglected, because the affinity of C5a(desArg) for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5a(desArg) to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5a(desArg) induce significant levels of cell activation that are even higher than those achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, because it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of G(α) proteins upon C5aR engagement by C5a or C5a(desArg). Further, mass spectrometric characterization of plasma-derived C5a and C5a(desArg) provided important insight into the posttranslational modification pattern of these anaphylatoxins, which includes glycosylation at Asn(64) and partial cysteinylation at Cys(27). Although the context-specific physiological contribution of C5a(desArg) has to be further explored, our data suggest that C5a(desArg) acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.