The effect of overexpression of endogenous delta-6 fatty acid desaturase gene (ER < DELTA > 6FAD) on eicosapentaenoic acid (EPA) production and total lipid content was investigated in Phaeodactyium tricornutum. All three randomly selected transformants exhibited significant increase (47.66%) in their EPA content, which reached up to 38.101 mg/g DW in algal strain D63. The total lipid content of all the three transformants was significantly higher (16.40-18.64%) than that of the wild-type strain. These findings suggested that the reaction catalyzed by ER < DELTA > 6FAD is a limiting step for EPA biosynthesis, overexpression of endogenous ER < DELTA > 6FAD gene is an effective method for improving EPA production in eukaryotic microalgae, and fatty acid metabolic pathway in microalgae can be genetically modified.