In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.