Macrophages are central in orchestrating the clearance of apoptotic cells and cellular debris during inflammation, with the mechanism(s) regulating this process remaining of interest. Herein, we found that the n-3 docosapentaenoic acid-derived protectin (PD_{n-3 DPA}) biosynthetic pathway regulated the differentiation of human monocytes, altering macrophage phenotype, efferocytosis, and bacterial phagocytosis. Using lipid mediator profiling, human primary cells and recombinant enzymes we found that human 15-lipoxygenases initiate the PD_{n-3 DPA} pathway catalyzing the formation of an allylic epoxide. The complete stereochemistry of this epoxide was determined using stereocontrolled total organic synthesis as 16S,17S-epoxy-7Z,10Z,12E,14E,19Z-docosapentaenoic acid (16S,17S-ePD_{n-3 DPA}). This intermediate was enzymatically converted by epoxide hydrolases to PD1_{n-3 DPA} and PD2_{n-3 DPA}, with epoxide hydrolase 2 converting 16S,17S-ePD_{n-3 DPA} to PD2_{n-3 DPA} in human monocytes. Taken together these results establish the PD_{n-3 DPA} biosynthetic pathway in human monocytes and macrophages and its role in regulating macrophage resolution responses.