Nacreous structure holds significant promise for biomimetic design applications due to its excellent mechanical properties. In this paper, the influence of loading direction on the mechanical properties of nacreous structure was investigated to understand its mechanisms of anisotropic fracture resistance. The results revealed significant anisotropy in the mechanical properties of nacre when loaded along different directions (perpendicular to y and z axes). When loaded perpendicular to the top face, nacre exhibits better mechanical properties and greater resistance to fracture compared to loading perpendicular to the side face. This distinction can be attributed to different cracking modes of organic interfaces in nacreous structure, where the nacreous structure loaded perpendicular to the top face exhibits more tortuous crack deflection, larger fracture stepped surface area and microcrack zones. Furthermore, the theoretical analysis indicates that the differences in the volume fraction of organic interfaces and the main crack deflection along organic interfaces are important factors leading to different cracking modes of organic interfaces in nacreous structure. Based on the analysis of the mechanisms of anisotropic fracture resistance, the critical parameters to characterize the anisotropic mechanical properties of the nacreous structure were proposed. The research results provide a reference and basis for future biomimetic application design.