Cholera toxin (CT) is an AB5‐subunit toxin that enters host cells by binding ganglioside GM1. GM1 carries the toxin retrograde from the apical cell surface through the trans‐Golgi network (TGN) into the endoplasmic reticulum, where a portion of the A‐subunit translocates to the cytosol to induce disease. In polarized cells that line mucosal surfaces, a fraction of the toxin enters the transcytotic pathway, moving all the way from apical to basolateral membrane, thus breeching the epithelial barrier. Here, we find that sorting of CT‐GM1 complexes into the transcytotic pathway occurs prior to delivery to the TGN or ER The closely related E. coli toxin LTIIb, which binds a different ganglioside, also enters the endosomal compartment of polarized cells but is not sorted into the retrograde or transcytotic pathways, suggesting that ganglioside structure may dictate the trafficking. To test this, we used fluor‐labeled GM1 species with defined ceramide structures applied to live cells. We found that polarized epithelia preferentially sort GM1 species with unsaturated ceramide acyl chains from apical to basolateral membranes by transcytosis. These results identify the early/common endosome as the site of GM1 sorting in polarized cells and show that trafficking depends on ceramide structure.