The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP⁺ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.