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Summary Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. InArabidopsis thalianaembryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between alteredISE2expression and intercellular trafficking.Gene expression analyses in Arabidopsis tissues whereISE2expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected.Concomitant with changes in the expression of GLS‐related genes, plants with abnormalISE2expression contained altered GLS metabolic profiles compared with wild‐type (WT) counterparts. Indeed, changes in the expression of GLS‐associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking.These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole‐type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity.