A feeding experiment was carried out to determine the effect of a diet lacking n-3 and n-6 highly unsaturated fatty acids (HUFA) on lipid and fatty acid content in intestine and gills of male gilthead seabream (Sparus aurata L.) broodstock at different stages of the reproductive cycle: November (pre-spermatogenesis), March (spermatogenesis), and June (post-spermatogenesis). For this purpose, gilthead seabream broodstock were fed either a control diet (C) or an n-3 and n-6 HUFA-deficient diet (D). The results showed no changes in fatty acid content of polar lipids of intestine and gills from fish fed diet C at different stages of the reproductive cycle. However, significant changes were observed in the fatty acid content of neutral lipids in intestine but not in gills in this group. Thus, between November and March, saturates and n-3 HUFA decreased while monoenes increased. In June, the contents of these fatty acids had returned to their initial values (November). Moreover, in fish fed diet D, the fatty acid content of neutral lipid changed in both intestine and gills. In intestine NL, a decrease in saturates and n-3 HUFA and an increment in monoenes were observed from November to June. In gills, a decrease was also observed in n-3 HUFA from NL along the cycle. Nevertheless, n-6 HUFA content remained unchanged. These results show both tissue specificity in seasonal mobilization of fatty acids linked to reproductive processes and the influence of dietary fatty acids on body composition.