This study evaluated the mechanical and ultrastructural changes during eggshell formation in laying hens by using the optimal levels of organic and inorganic manganese (Mn). A total of 270 62-wk-old Hy-Line Brown laying hens were fed a basal diet containing 25.1 mg Mn per kg feed for 2 wks, after which they were randomly allocated into 3 groups and fed the basal diet (control) or the basal diet supplemented with 120 mg Mn per kg feed from monohydrate Mn sulfate (an inorganic source of Mn), or 80 mg Mn per kg feed from an amino acid-Mn complex (an organic source of Mn) for 12 wks. For each group, 6 replicates of 15 hens were used with one hen per cage. Dietary Mn supplementation significantly increased eggshell-breaking strength and thickness in laying hens (P < 0.05). In neither was the elasticity of their eggshell membranes, measured during the nucleation and mammillary knob formation stages, affected by dietary Mn compared with the control (P > 0.05), whereas the breaking strength of the eggshells was greater at the linear and terminate deposition stages compared with the control (P < 0.05). Dietary Mn supplementation decreased the width of the mammillary knobs and the proportion of mammillary thickness, and increased the proportion of effective thickness of the whole eggshells (P < 0.05). Ultrastructural changes during the eggshell formation indicated that dietary Mn supplementation increased the nucleation site and mammillary knob densities, decreased the mammillary thickness, and increased the proportion of effective thickness and total thickness of the eggshells compared with the control (P < 0.05). Therefore, dietary Mn supplementation can improve the breaking strength and ultrastructure of the eggshells during their formation, and the mammillary and palisade layers are both crucial structures affected by Mn.