Mollusk shells generally consist of several macrolayers with different microstructures. To explore the specific role that different macro-layers play in the overall mechanical properties of shells, the microstructures, hardness distribution, and three-point bending behavior in the deep-sea Nautilus shell were investigated. It is found that the shell presents a hierarchical structure comprising three layers in thickness, that is, the outer, middle, and inner layers, which exhibit homogeneous, prismatic, and nacreous structures, respectively. Among them, the homogeneous structure in the outer layer is harder, which is beneficial for the shell to enhance resistance to wear and perforation. Furthermore, both the bending strength and fracture energy for group Up (loading from outer to inner surfaces) are far higher than those for group Down (loading from inner to outer surfaces), indicating that the inner nacreous layer is not only stronger but also tougher. Cracks tend to deflect at the interfaces in nacreous structure, and nacreous structure is thereby more resistant to breakage. Hence, the nacreous structure in the inner layer could protect the shell from breaking catastrophically in the deep sea with high pressure. In brief, the combination of a harder outside layer and a tougher inside layer provides an effective protective structure for the deep-sea shell, and the excellent environment adaptability of Nautilus shell can thus be interpreted in terms of its ingenious microstructure arrangement.