Since the discovery of graphene, two-dimensional nanosheets have attracted attention for their unique shapes and properties. The simplest production method is the top-down approach, in which layered materials are mechanically cleaved or exfoliated. However, preparing biomaterial nanosheets by the top-down method is challenging, and to date, only a few reports have been published. In this study, we developed native cellulose nanosheets with a thickness of several tens of nanometers, which maintained the native uniaxial orientation of cellulose fibers from wood cell walls through a combination of mild delignification and mild homogenization. The wood cell wall is a complex structure composed of three main polymers, cellulose, hemicellulose, and lignin. The elementary unit of wood cellulose is a fiber 3–4 nm in width, which exists as bundles within the cell wall. Although cellulose has been disintegrated into nanofibers in various ways, cellulose nanosheets have not previously been isolated from wood cell walls. Nanocellulose is a developing material for various applications, taking advantage of its lightweight nature, high strength, excellent thermal dimensional stability, and superior gas barrier properties. A wide variety of performances can be achieved by controlling its morphology. The resulting nanosheets preserved the native orientation of the cellulose microfibrils in the cell walls, providing evidence in support of the presence of a lamellar cellulose structure within the cell wall.