The bioaccessibility of total lipids and fatty acids (FA) in raw and grilled gilthead seabream (Sparus aurata) was determined using an in vitro digestion model. The particular impact of grilling on the FA profile of seabream was also studied. In addition, the influence of lipid class on the bioaccessibility of each FA was analysed. Grilling did not change the relative FA profile, and only the absolute values were altered. However, the relative FA profile varied across lipid classes, being more dissimilar between TAG and phospholipids. Long-chain SFA and PUFA seemed to be less bioaccessible. Moreover, grilling reduced bioaccessibility of protein, fat and many FA, with the highest reductions found in PUFA such as the DHA. Strong evidence supporting a predominantly regioselective action of lipase during in vitro digestion was found, and the impact of this phenomenon on FA bioaccessibility was assessed.