Taking Nanjing Yangtze River Shield Tunnel with large cross-section (Φ14.5m) as background, the mechanical characteristic of lining structure under high hydrostatic water pressure for different segmental assembling modes is studied by large-scale similar model test. The test system can control earth pressure, uniform and non-uniform water pressure acting on lining respectively and cooperatively. The study results show that the values and distribution of deformation and internal force vary with different assembling modes. The rigidity of the whole ring for straight joint assembling is less, inducing the deformation larger and internal force smaller, which is unfavorable for waterproofing requirements. The peak positions of bending moment change with the difference of staggered joint assembling modes. Due to the smaller rigidity of K segment, the value of bending moment on that position is less. The most favorable and unfavorable assembling modes are proposed for construction design for Nanjing Yangtze River Shield Tunnel; meanwhile it can also provide important reference to the correlative project.