The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification–untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7( S )-hydroxy-docosahexaenoic acid [7( S )-HDHA]. Results from mass spectrometric detection of 7( S )-HDHA in mouse and rat brain tissues, time-resolved FRET analyses, and thermal shift assays collectively revealed that 7( S )-HDHA potently activated PPARα with an affinity greater than that of other ligands identified to date. We also found that 7( S )-HDHA activation of PPARα in cultured mouse cortical neurons stimulated neuronal growth and arborization, as well as the expression of genes associated with synaptic plasticity. The findings suggest that this DHA derivative supports and enhances neuronal synaptic capacity in the brain.