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Abstract Background The Diplolepideae are the larger group within the Arthrodontae mosses, characterized by peristomes formed from residual cell walls. It is now understood that these peristomes exhibit diverse hygroscopic movements, playing a crucial role in spore release. However, the exact mechanism behind this movement remains unclear, lacking direct evidence. This study investigated the microscopic and submicroscopic structures of the peristomes in three Diplolepideae species: Hypopterygium fauriei (Besch.), Pylaisia levieri (Müll. Hal.) Arikawa and Regmatodon declinatus (Hook.) Brid. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to reveal the differences in their hygroscopic movement mechanisms. Results The three species exhibited distinct responses upon wetting: H. fauriei’s exostome closed inwards, P. levieri’ opened outwards, and R. declinatus’ elongated significantly. These differences are attributed to the varying microfibril deposition in the exostome layers. Uniform deposition in the inner layer and minimal deposition in the outer layer enabled exostome opening upon wetting and closing when dry. Our findings suggest that the diastole and contraction of fine microfibrils in the exostome plates and ridges are the key drivers of hygroscopic movement. Conclusions This study provides further evidence at both the structural and submicroscopic levels, contributing to the unraveling of the hygroscopic movement mechanism in Diplolepideae peristomes. This enhanced understanding sheds light on the relationship between peristome structure and function.