Sea cucumbers Holothuria atra and Holothuria leucospilota play an important role in the bioturbation of sediment in coral reef and rocky intertidal ecosystems. This study investigated changes in sediment fatty acid (FA) composition during gut passage in H. atra and H. leucospilota. The FA composition did not differ significantly between species. Comparison of FA composition in ambient sediment (AS), foregut (FG), midgut (MG), hindgut (HG), and faecal pellets (FPs) indicated that marked changes in FA composition occurred during passage through the gut of H. atra and H. leucospilota. Saturated fatty acids (SAFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and branched fatty acids (BrFAs) were significantly higher in FG than in AS, suggesting that both species selectively ingested nutrient rich particles. Significant reduction of SAFAs, MUFAs, PUFAs, and BrFAs occurred in MD and HD, with complete elimination of most PUFAs in FPs. A decrease in PUFAs 20:5 ω 3, 18:4 ω 3, 22:5 ω 3, 22:6 ω 3, 18:2 ω 6, 18:3 ω 3, 18:3 ω 6, odd-numbered BrFAs, and MUFA 18:1 ω 7 indicated that algal detritus and bacteria were important part of diet. These results have implications for the fate of specific dietary FAs, especially ω 3 and ω 6, and the contribution holothurian FPs make to the FA composition of coral reef and rocky intertidal ecosystems.