The degree of fatty acid unsaturation in membrane phospholipids affects many membrane‐associated functions and can be influenced by dietary consumption of fatty acids such as saturated fatty acids and polyunsaturated fatty acids (PUFAs). Cells must adapt to changes in composition of membrane fatty acids by regulating lipid‐metabolizing enzymes. In this study, we investigated how cells respond to loading with excess PUFAs, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. A lipidomics analysis revealed that dipalmitoylphosphatidylcholine (DPPC) was increased after the production of PUFA‐containing phospholipids in cells loaded with PUFAs. An RNA interference screen of lipid‐metabolizing enzymes revealed that lysophosphatidylcholine acyltransferase 1 (LPCAT1) was involved in the DPPC production. Moreover, LPCAT1 knockdown markedly enhanced the cytotoxicity induced by excess PUFAs. PUFA‐induced cytotoxicity was dependent on caspase and unfolded protein response (UPR) sensor proteins inositol requiring 1a and protein kinase R‐like endoplasmic reticulum kinase, suggesting that excess PUFAs trigger UPR‐mediated apoptosis. In murine retina, in which PUFAs are highly enriched, DPPC was produced along with increase of PUFA‐containing phospholipids. In LPCAT1 knockout mice, DPPC level was reduced and UPR was activated in the retina. Our results provide insight into understanding of the retinal degeneration seen in rd11 mice that lack LPCAT1.—Akagi, S., Kono, N., Ariyama, H., Shindou, H., Shimizu, T., Arai, H. Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids. FASEB J. 30, 2027–2039 (2016). www.fasebj.org