Crypthecodinium cohnii, a protist renowned for its high docosahexaenoic acid (DHA) production, has an unclear mechanism for converting docosapentaenoic acid (DPA) into DHA. This study employed transcriptomic analysis to investigate the effect of excessive oxygen limitation (EOL) on DHA biosynthesis, uncovering a novel oxygen-dependent pathway. The use of intermittent oxygen limitation (IOL) strategy significantly boosted DHA production. Five △4-fatty acid desaturase (FAD4) genes were identified, with CcFAD4_52534 exhibiting the highest catalytic efficiency and dual-functionality, converting eicosapentaenoic acid (EPA) to DPA, and subsequently to DHA. This study integrates transcriptomic insights, the discovery of the bifunctional CcFAD4_52534 enzyme, and the optimized IOL strategy, offering transformative potential for sustainable and high-yield DHA production through tailored genetic engineering in C. cohnii. This approach bridges ecological understanding with industrial innovation.