The original strain Pythium was mutated by UV combined with LiCl.After preliminary screening by low-temperature culturing and microscope observation after Sudan Ⅱ staining,and re-screening by shake flask fermentation,a high-yielding eicosapentaenoic acid-producing strain Pythium H6 was obtained.Its biomass,oil and EPA content was 23.5 g /L,17.54 % and 15.36 % respectively after two stages of 15~20℃ culture for 168h.In addition,the EPA production of H6 was 633.17 mg/L,which was 140.01 % higher than the original strain,and the product character was stable.The study of the lipid synthesis of Pythium H6 exposed to different concentrations of exogenous citric acid indicated that adding 1.5 g/L citric acid to fermentation medium can significant increase its glucose utilization,lipid and EPA content with EPA yield up to 890.49 mg/L,40.64 % higher compared to the control.Fermentation experiments suggested that Pythium H6 has the potential to be developed into a highly industrial production strain.