The role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca(2+)-dependent membrane-binding protein Annexin B9 (AnxB9), apical β(Heavy)-spectrin (β(H)) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of β(H) spliceoforms, and loss of AnxB9 results in an increase in basolateral β(H) and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when β(H)-positive endosomes are generated either by upregulation of β(H) in pak mutants or through the expression of the dominant-negative version of β(H). In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or β(H). Loss of AnxB9 or β(H) function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes β(H)) mutants. Reduction of AnxB9 results in degradation of the apical-lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to β(H). These results indicate that AnxB9 and β(H) are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.