P-glycoprotein (P-gp) is mainly found in the cell membrane of the small intestine and blood-brain barrier in vivo, and is responsible for the extracellular transport of cytotoxic hydrophobic compounds. P-gp is known to transport many pharmaceutical compounds as substrates. If we can understand the substrate recognition mechanism of P-gp, it will be possible to design pharmaceutical compounds that are not recognized by P-gp. Recently, the complex structures of human P-gp with substrates and inhibitors have been reported by single-particle analysis using Cryo-EM, and the differences in the binding pockets of substrates and inhibitors have been clarified. However, a detailed understanding of how P-gp can identify compounds as substrates or inhibitors has not been achieved. In this study, we aim to elucidate the detailed substrate recognition mechanism by elucidating and comparing multiple complex structures of P-gp and compounds. First, we established a system for expression and purification of human P-gp. Further, we have established a simple system for reconstitution into Nanodisc. Recently, we succeeded to obtain the 3D structure at the highest resolution (2.93 Å) as human P-gp. In this presentation, we will introduce the expression, purification, and Nanodisc reconstruction systems of P-gp and the obtained 3D structures.