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Eicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, <i>Mucor circinelloides</i>, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, <i>M. circinelloides</i> lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). <i>M. circinelloides</i> could produce DGLA by overexpressing the <i>D6E(GLELO)</i> gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (<i>PpD17</i> and <i>PaD17</i>) genes from <i>Phytophthora parasitica</i> and <i>Pythium aphanidermatum</i>, respectively, were introduced into <i>M. circinelloides</i> to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains’ biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of <i>cme2</i>, <i>fas2</i>, and <i>D6E</i>, thereby contributing to lipid biosynthesis in <i>M. circinelloides</i>. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in <i>PpD17</i> overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the <i>PpD17</i> gene in <i>M. circinelloides</i>, which established a new scope for further research in the production of ETA in oleaginous fungi.