Sea cucumber Apostichopus japonicus is one species of deposit feeders with abundant food resources. The analysis of fatty acid biomarkers provides an alternative method of investigating diets, which is not subject to the biases of stomach content analysis. In the present study, 16:1(n-7)/16:0 and EPA ［20:5(n-3)］ were selected as diatom fatty acid biomarkers according to the fatty acid composition in body wall tissues of A. japonicus. DHA ［22:6(n-3)］, ∑［18:2(n-6)+18:3(n-3)］ and 20:4(n-6) were used as fatty acid biomarkers of flagellate or protozoan alga, Chlorophyta and brown alga respectively; Odd & branched FAs and 18:1(n-7) were the fatty acid biomarkers of heterotrophic bacteria. Seasonal variations of food sources in sea cucumber A. japonicus, sampled from a critical cofferdam aquaculture pond, were determined by fatty acid biomarkers analysis. The results showed the food sources consisted of diatoms, flagellate or protozoan, bacteria, brown alga and Chlorophyta originated organic matters. The sea cucumber A. japonicus was characterized by significant diatom markers such as high ratio of 16:1(n-7)/16:0 (0.731.82) and EPA contents (7.24%-14.45%), so diatoms were speculated as one of the main diets. And the two diatom markers showed significant variations during the period the experiments (F=8.871, P<0.001; F=22.882, P<0.001; respectively). The low contents of DHA (2.41%-4.45%) and DHA/EPA ratio (0.29-0.40) showed the flagellate or protozoan provided low proportions of diets for A. japonicus in the year. The food contribution of Chlorophyta was also low with the biomarker ［∑18:2(n-6)+18:3(n-3)］ changing from 1.51% to 2.28% in the duration of the study. However, the relative contents of brown seaweed biomarker 20:4(n-6) were also found high in the year (4.88%-8.16%) with the peak values appearing in autumn and winter. Fatty acid biomarkers specific to bacteria CytophagaFlavobacteria and Proteobacteria were also found in considerable amounts in the sea cucumber tissue, which suggested that there were substantial bacterial input into the food of the species. The principal component analysis showed the main food sources of A. japonicus changed with seasons. Diatoms, flagellate or protozoan, brown alga and bacteria were the main diets of the sea cucumber in January. The contribution of diatoms, flagellate or protozoan and Chlorophyta were comparatively high in March. Chlorophyta was the most important diets for A. japonicus in June. The diets of sea cucumber were mainly bacteria and Chlorophyta in July. Bacteria were the most important food source of A. japonicus in August and September, and the brown alga and bacteria supplied considerable amounts of food for the sea cucumber from October to November. The present study provides alternative materials for feed research of sea cucumber A. japonicus.