Mitochondria is double-membrane bound organelles and fuse each other to regulate the shape, size and fundamental function. Optic atrophy 1 (OPA1), 960-residue membrane protein is required for mitochondrial inner membrane fusion. As previously discussed, reconstitution of membrane fusion with recombinant protein and model membrane have been utilized for elucidating the detailed molecular mechanism of membrane fusion. However, reconstituting approach of mitochondrial inner membrane fusion have not been reported, because preparation of relatively large mammalian membrane proteins is often difficult, mainly due to the low stability and high propensity to aggregation when expressed by bacterial expression system. In this chapter, we describe an advance in the expression and purification of OPA1 by the bacmid-silkworm expression system. We also show reconstitution of OPA1-mediated in vitro membrane fusion, and the unique fusion machinery of mitochondrial inner membrane fusion. Because the bacmid-silkworm expression system might have advantages for the expression of large membrane proteins, the present method will have general applicability to various mitochondrial membrane proteins.