OBJECTIVE Polyunsaturated fatty acids (PUFA) are a source of bioactive lipids regulating inflammation and its resolution. METHODS Changes in PUFA metabolism were compared between lacrimal glands (LGs) from young and aged C57BL/6J mice using a targeted lipidomics assay, as was the gene expression of enzymes involved in the metabolism of these lipids. RESULTS Global reduction in PUFAs and their metabolites was observed in aged LGs compared to young controls, averaging between 25-66% across all analytes. ꞷ-6 arachidonic acid (AA) metabolites were all reduced in aged LGs, where the changes in prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) were statistically significant. Several other 5-lipoxygenase (5-LOX) mediated metabolites were significantly reduced in the aged LGs, including D-series resolvins (e.g., RvD4, RvD5, and RvD6). Along with the RvDs, several ꞷ-3 docosahexaenoic acid (DHA) metabolites such as 14-HDHA, neuroprotectin D1 (NPD1), Maresin 2 (MaR2), and MaR1 metabolite (22-COOH-MaR1) were significantly reduced in aged LGs. Similarly, ꞷ-3 eicosapentaenoic acid (EPA) and its metabolites were significantly reduced in aged LGs, where the most significantly reduced was 18-HEPE. Using metabolite ratios (product:precursor) for specific metabolic conversions as surrogate enzymatic measures reduced 12-LOX activity was identified in aged LGs. CONCLUSIONS In this study, global reduction of PUFAs and their metabolites was found in the LGs of aged female C57BL/6J compared to young controls. A consistent reduction was observed across all detected lipid analytes except for ꞷ-3 docosapentaenoic acid (DPA) and its special pro-resolving mediators (SPM) in aged mice, suggesting an increased risk for LG inflammation.