A growing body of evidence indicates a link between circulating neurotoxic lipids and the development of chronic neuroinflammatory diseases in the peripheral and central nervous systems. Therefore, strategies to modify circulating lipid profiles may complement the management of neuroinflammatory diseases, including neuropathic pain. In a previous study, we observed a sig-nificant shift in the metabolomic profile of patients' plasma with symptoms of painful diabetic neuropathy (pDN) following three months of docosahexaenoic acid (DHA)-rich supplementation, leading to improved pDN symptoms. However, it is important to identify the specific lipid mediators responsible for this therapeutic effect and elucidate potential mechanism(s). This study investigates whether DHA-rich supplementation reduces neurotoxic lipid mediators associated with pDN in individuals with type 2 diabetes mellitus (T2DM). Forty volunteers diagnosed with type 2 diabetes were enrolled in the "En Balance-PLUS" diabetes education study. The volunteers participated in weekly lifestyle/nutrition education and daily supplementation with 1000 mg DHA and 200 mg eicosapentaenoic acid. The Short-Form McGill Pain Questionnaire validated the clinical determination of baseline and post-intervention pain complaints. Untargeted Lipidomic analyses were conducted using blood serum collected at baseline and after three months of participation in the dietary regimen. The lipidomic data were analyzed using a non-parametric paired Wilcoxon rank-sum test and random forest analysis. ELISA further eval-uated participant serum samples to investigate associated biomarkers of necrosis (MLKL), autophagy (ATG5), and lipid chaperone protein (FABP5). Untargeted lipidomic analysis revealed that several neurotoxic-associated lipids significantly decreased after DHA-rich supplementation. Also, circulating levels of MLKL were reduced, while protein levels of ATG5 and FABP5 significantly increased. The reduction of circulating neurotoxic lipids and increase of neuroprotective lipids following DHA-rich supplementation is consistent with the reported roles of omega-3 polyunsaturated fatty acids (PUFAs) in reducing adverse symptoms associated with neuroinflammatory diseases and painful neuropathy.