The aim of this study was to evaluate the effect of a dietary highly unsaturated fatty acid (HUFA) deficiency on winter flounder Pseudopleuronectes americanus metamorphosis by examining the growth and the expression of genes involved in some key metabolic processes: lipid digestion, oxidative stress, and growth. Three groups of fish were fed rotifers enriched with different blends of microalgae providing different HUFA profiles: (1) a diet comprising a mixture of three microalgae, Nannochloropsis oculata, Isochrysis galbana, and Pavlova lutheri (Cocktail diet), which contained a balanced combination of ecosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA); (2) the N. oculata diet (nanno diet), with a low level of DHA; and (3) the I. galbana diet (Tiso diet), characterized by low levels of EPA and AA. The results indicate that the need for DHA increased from settlement and for EPA and aa from 15 days after settlement. The lower HUFA content in the Tiso and nanno diets did not affect larval development or lipid reserve accumulation. The superoxide dismutase gene expression suggests a reduced oxidative stress in the Cocktail group, and overall results indicate that gh gene expression could be a valuable indicator of development at the molecular level in response to dietary HUFA quality during metamorphosis in winter flounder.