Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined byβ-1,3-glucan synthase versus glucanase activities. A proteomic analysis of anArabidopsis thalianaplasmodesmata (Pd)-rich fraction identified aβ-1,3-glucanase as present in this fraction. The protein encoded by theputative plasmodesmal associated protein(ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) orNicotiana benthamianaepidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane markerN-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4–64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribeAtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.