This study evaluated the mechanism by which dietary manganese (Mn) supplementation-in either an organic or inorganic form-affects mammillary knobs of the eggshell ultrastructure in laying hens. A total of 225 54-week-old Hy-Line Brown laying hens were fed a basal diet containing 27.5 mg Mn/kg feed for 2 wk, after which they were randomly allocated into 3 groups and fed a basal diet (control) or a basal diet supplemented with 120 mg Mn/kg feed from monohydrate Mn sulfate (an inorganic source of Mn) or with 80 mg Mn/kg feed from an amino acid-Mn complex (an organic source of Mn) for 10 wk. For each group, 5 replicates of 15 hens each were used with 1 hen per cage. Compared with the control, dietary Mn supplementation increased the mammillary-knob density of eggs at 9.5 h post-oviposition (P < 0.05). The Mn content in both blood and eggshell gland was increased with the supplementation of Mn in inorganic and organic forms (P < 0.05), but the blood Mn content was higher after inorganic-Mn supplementation as compared with organic-Mn supplementation (P < 0.05). RNA sequencing and quantitative real-time PCR analysis of the eggshell gland showed that dietary Mn supplementation increased the expression of genes encoding some proteoglycans, glycoproteins, and calcium-binding proteins in the eggshell gland (P < 0.05), and involved in the process of the protein glycosylation and glycan metabolism in the eggshell gland (P < 0.05). Overall, dietary Mn supplementation can involve in the process of protein glycosylation and glycan metabolism and improve the expression of genes encoding proteoglycans and glycoproteins in the eggshell gland, thus increasing the mammillary-knob density during the initial deposition stage of shell formation.