Regulation of leukocyte activation is critical to limit unintended tissue injury during acute inflammation. On neutrophil activation, polyisoprenyl diphosphate phosphatase 1 (PDP1) rapidly converts presqualene diphosphate to presqualene monophosphate to facilitate cell activation. Lipoxins are potent anti-inflammatory mediators for neutrophils, yet their counterregulatory signaling mechanisms remain to be determined. 15-Epi-lipoxin A 4 (15-epi-LXA 4) blocked agonist-initiated association of the nicotinamide adenine dinucleotide phosphate oxidase components p47 PHOX and p22 PHOX in human neutrophils. 15-Epi-LXA 4 (0.1–100 nM) inhibited neutrophil superoxide anion (O 2−) generation in a concentration-and ALX/FPR2 receptor-dependent manner that was disrupted by PDP1-specific antibodies. In differentiated HL60 cells, a myeloid cell line, agonist-initiated O 2− generation was inhibited by PDP1 siRNA. Recombinant human PDP1 was directly phosphorylated in vitro by select protein kinase C (PKC) isoforms, including PKCβII. When neutrophils were exposed to formyl-methionyl-leucyl-phenylalanine (fMLP), PKCβII was rapidly phosphorylated and physically associated with PDP1. Agonist-initiated conversion of neutrophil presqualene diphosphate to presqualene monophosphate was blocked by PKCβII inhibition. Neutrophil exposure to 15-epi-LXA 4 attenuated fMLP triggered PKCβII phosphorylation and its interactions with PDP1. Together, these findings indicate that PDP1 serves an integral signaling role in neutrophil proinflammatory responses and as a target for counter-regulatory mediators.—Carlo, T., Kalwa, H., Levy, BD 15-Epi-lipoxin A 4 inhibits human neutrophil superoxide anion generation by regulating polyisoprenyl diphosphate phosphatase 1.