In this study, the gene of a novel lipase with sn-1(3) regioselectivity (i.e., sn-1 or sn-3 specific) from Cordyceps militaris was successfully expressed by a heterologous expression system. Total RNA was extracted from C. militaris and then single-stranded cDNA was synthesized. The resulting C. militaris lipase (CML) gene was inserted in Escherichia coli expression plasmids [pET-296( +), pET-26b, and pColdIII] to construct plasmids encoding CML, which were then transformed to E. coli strains BL21 (DE3), C43 (DE), C41 (DE3), and Origami (DE3) for protein expression. Although the recombinant CML expression level was high, it was overproduced in the form of inclusion bodies. Under a specific condition, the soluble form of the recombinant CML was detected using Western blot analysis; however, no enzyme activity was observed. To overcome the lack of post-translational modifications in recombinant CML, a baculovirus-insect expression system was introduced for eukaryotic lipase expression. pDualBac was used as the transfer vector, and the CML gene was fused under the control of the polyhedrin promoter. After generating the recombinant baculovirus, the active form of CML was successfully produced and its kinetic parameters were determined.