Polyunsaturated fatty acids (PUFAs) have important industrial， physiological， and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1Δ9 to the important PUFA， 18:3Δ9，12，15， whereas the fungus Fusarium verticillioides 7600 uses the bifunctional desaturase Fm1 for both reactions. Here， we used a combination of sequence comparisons， structural modeling， and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu-153， Phe-157， and His-194)， located near the catalytic iron ions， predominantly affects activity， whereas cluster II (Tyr-280， His-284， and Leu-287)， located in a helix forming the entrance to the substrate-binding pocket， mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L， H284V， and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate， but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates， converting 16:1Δ9 to 16:3Δ9，12，15， consistent with Fm1 performing sequential v+3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate head group when the acyl chain contains both Δ9 and Δ12 double bonds， in which case C15 becomes positioned adjacent to the di-iron site enabling a second v+3 desaturation.