BackgroundThis study aimed to explore the mechanism of organic zinc and manganese (OZM) influencing eggshell quality of laying hens. A total of 384 21‐week‐old Jingfen‐1 laying hens were randomly divided into four groups (8 replicates per group, 12 hens per replicate): control group (60 mg kg−1 Zn as zinc sulfate and 80 mg kg−1 Mn as manganese sulfate) and OZM groups (5, 10 and 15 mg kg−1 Zn as yeast zinc and 80 mg kg−1 Mn as manganese methionine).ResultsCompared with the control group, OZM_5 and OZM_10 groups significantly increased feed intake and egg weight in laying hens in 28–31 weeks (P < 0.05) while egg weight and shell weight were increased in all the OZM groups (P < 0.05). In addition, the OZM_15 group significantly decreased the mammillary knob width and mammillary layer thickness and increased the palisade layer thickness and total effective thickness (P < 0.05), and had a stronger and more stable Ca signal strength in the mammillary layer and a more uniform and dense distribution of calcium and zinc in the transversal surfaces of eggshell. Furthermore, mRNA levels of Claudin2, Zona Occludens 1, Alkaline phosphatase and Ca2+ATPase were significantly upregulated in the OZM_15 group of laying hens at week 31 (P < 0.05).ConclusionDietary supplementation of 10–15 mg kg−1 yeast zinc and 80 mg kg−1 manganese methionine can enhance eggshell ultrastructure by influencing its endometrial barrier function and calcium deposition, thereby improving eggshell quality. © 2024 Society of Chemical Industry.