The Vitreoscilla hemoglobin gene was expressed in Pythium to alleviate oxygen limitation and improve the yields of eicosapentaenoic acid during fermentation.Furthermore,the growth characteristic of recombinant strain was studied.We constructed an expression vector containing hemoglobin gene and transformed it into Pythium through the mediation of Agrobacterium LBA4404.We evaluated the effect of hemoglobin gene on growth of Pythium and biosynthesis of eicosapentaenoic acid in shake flasks.The results showed that the Pythium V1 containing hemoglobin gene was obtained and the hemoglobin gene was successfully expressed.Through the optimization of culture conditions,the biomass,lipid in mycelium and content of eicosapentaenoic acid in lipid of Pythium V1 respectively increased 4.8%,4.82% and 11.69% compared with the parental strain in shake flasks,and the yields of EPA was 1 004.96 mg/L.The expression of hemoglobin gene in Pythium could improve the yields of eicosapentaenoic acid,thus it showed a good application prospect to solve the problem about the low dissolved oxygen in high density fermentation of the filamentous fungi.