Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes.