Abstract: Mechanosensitive ion channels are essential for sensing and converting mechanical forces into electrical or chemical signals. These channels are widely distributed across bacteria, animals, and plants. In Arabidopsis thaliana, the OSCA family has been identified as mechanically activated ion channels that respond to osmotic stress by allowing calcium ions to enter the cell. This influx increases the cytoplasmic calcium concentration, triggering osmotic stress-induced signal transduction cascades in plants. In this study, we determined the structures of OSCA2.2 and OSCA3.1 via cryoelectron microscopy (cryo-EM). Both proteins form homodimers consisting of 11 transmembrane helices (TM1–11). The ion conduction pathway is formed by TM4–8. Despite belonging to the same family, OSCA2.2 and OSCA3.1 exhibit notable structural variations. Structural analysis revealed that both OSCA2.2 and OSCA3.1 exhibit a closed conformation. We also conducted functional studies on OSCA proteins via electrophysiological experiments and confirmed the role of key amino acids in the process of ion permeation.