A 15 days feeding experiment was carried out to investigate the requirement for essential fatty acids (EFA) of the juvenile puffer fish Takifugu rubripes, using Artemia nauplii enriched with various types of different fatty acids. Experiment was conducted under laboratory condition and ambient water temperature. There was no significant difference in the growth among the juveniles fed on Artemia enriched with oleic acid (OA), linolenic acid (LNA) and eicosapentaenoic acid (EPA) alone. Whereas, the results obtained show that docosahexaenoic acid (DHA) alone or n-3 highly unsaturated fatty acids (n-3 HUFA) were much more effective as EFA for juvenile puffer fish to achieve the best feeding performance. On the other hand, final survival rate and activity test of juvenile puffer fish were not significantly different among all of the groups during the experimental period. The best results, however, were obtained in the groups fed on Artemia containing n-3 HUFA rather than enriched with fatty acids of either EPA alone or DHA alone. These results suggest that the juvenile puffer fish required both EPA and DHA as their EFA, although DHA was more effective than EPA.