α-Synuclein (α-syn) is the major constituent of Lewy bodies and glial cytoplasmic inclusions which are pathological hallmarks of neurodegenerative disorders like Parkinson's disease or multiple system atrophy (MSA), respectively. It accumulates and aggregates during the pathogenic process, and missense mutations, such as A53T, are increasing its probability of aggregate formation. Furthermore, α-syn interacts with polyunsaturated fatty acids, and this interaction may promote the oligomerization process. To investigate whether membrane lipid modification by docosahexaenoic acid (DHA) modifies the aggregation process of α-syn in oligodendroglial cells, we have used OLN-93 cells stably expressing the human α-syn A53T mutation. Cells were supplemented with DHA (25 μM) for 3 days and then subjected to oxidative stress (OS) exerted by hydrogen peroxide. The data show that modification of the oligodendroglial cell membranes by DHA followed by OS caused the formation of fibrillary α-syn inclusions, a decrease in α-syn solubility, and an increase in phosphorylation at serine 129, which has been suggested to play a proaggregatory role. The aggregates contain αB-crystallin and ubiquitinated proteins and SUMO-1 immunoreactivity. SUMO-1 has been implicated in protein aggregation and identified as a constituent in inclusion bodies in MSA. Hence, membrane lipid modification in oligodendroglial cells promotes the formation of α-syn inclusion bodies resembling protein aggregates in neurodegenerative disease. This effect is not only attributable to the A53T mutation but also is observable in OLN cells expressing wild-type α-syn.