In order to increase omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) availability and to improve the design of rotifer's enrichment protocols for marine larvae production, the endogenous fatty acid (FA) metabolism of the marine rotifer, Brachionus plicatilis was determined. To this purpose, the in vivo ability of this species to incorporate, esterify into the different lipid classes and to elongate/desaturate unsaturated FAs was investigated. Rotifers were incubated in 75 cm2 tissue culture flasks, at a density of 75,000 rotifers per incubation, for 4 h. Incubations (n = 4) were performed at 21 °C, with 0.3 μCi of [1-14C]FAs including C18 FAs (18:1n-9, OA; 18:2n-6, LA; 18:3n-3, ALA) and LC-PUFA (20:4n-6, ARA; 20:5n-3, EPA; 22:6n-3, DHA). The present study demonstrates that rotifers possess an active metabolism over dietary FAs, being this species able to produce LC-PUFA from its C18 FA precursors. Interestingly, the action of a Δ12 desaturase that converts OA to LA and of a Δ15 desaturase over LA to produce ALA, seems to be also present, in agreement with previously reported functional characterization of ωx desaturases genes in this species. However, a globally low transformation rate was obtained, in addition to a lower incorporation of DHA into rotifer tissues compared to the other incubated substrates. The aforementioned factors, together with a competitive esterification pattern of DHA, LA and ALA into phosphatidylcholine (PC) or of EPA and ARA into phosphatidylinositol (PI), may give rise to rotifers with inadequate essential FA ratios for marine larvae nutrition, if ignored or incorrectly handled in the lipid enrichment process of this live prey species.