This is the first comprehensive study on the effect of dietary polyunsaturated fatty acid (PUFA) levels on the expression of fatty acid elongase 5 (AJELOVL5), PUFA composition, and growth in juvenile sea cucumbers. The specific growth rate (SGRw) was improved in n-3 PUFA-rich diets compared to low n-3 PUFA diets. AJELOVL5 expression was apparently upregulated in juveniles fed lower PUFA diets relative to higher PUFA diets, with higher expression in the body wall and respiratory tree of juveniles fed diets without ɑ-linolenic acid (ALA, 18:3n-3) compared to juveniles fed higher ALA level diets; similar results were also detected in juveniles fed diets with lower eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and none of ALA, EPA, or DHA respectively. The concentrations of ALA, EPA, and DHA in tissues were positively related to the content of dietary corresponding PUFA, with higher ALA content in juveniles fed diet ALA12.71 than in the ALA7.46 and ALA0 groups. Similar results were also obtained in sea cucumber fed diets enriched with either EPA or DHA. Interestingly, considerable levels of EPA and DHA were found in the tissues of juveniles fed diets of CK0 and DHA0, with no specific input of EPA or DHA, showing that the sea cucumber was capable of biosynthesizing EPA and DHA from their corresponding precursors as ALA and linoleic acid (LA, 18:2n-6).