Protectin D1 [neuroprotectin D1 (NPD1), PD1] has been proposed to play a key role in the resolution of inflammation. Aside from its ω-monohydroxylated metabolite, little has been reported on its metabolic fate. Upon NPD1 incubation in HepG2 cells, liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the formation of two main metabolites, identified as 2,3-dinor-NPD1 and 2,3,4,5-tetranor-NPD1 by comparison with standards obtained through demanding total chemical syntheses. These data represent the first evidence of β-oxidation occurring in specialized proresolving mediators and show that the biotransformation of NPD1 by human hepatoma cells is extremely rapid and faster than that of leukotriene (LTE4). Unlike LTE4, the main metabolic process occurs from the polar head chain of NPD1. It may limit NPD1 systemic circulation and prevent its urinary excretion, making difficult its detection and quantitation in vivo. Interestingly, tetranor-NPD1, but not dinor-NPD1, maintained the bioactivity of the parent NPD1, inhibiting neutrophil chemotaxis in vitro and neutrophil tissue infiltration in vivo.