Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/β-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D. The Wnt signaling pathway controls diverse biological processes such as growth, patterning and differentiation. Misregulation of the Wnt pathway can lead to human diseases including cancer and metabolic disorders 1-3. Wnt signaling is evolutionarily conserved, and hence Drosophila has been extensively utilized for genetic dissection of the Wnt signal transduction pathway. Secreted Wingless (Wg, Drosophila Wnt1) activates a cascade of signal transduction events. Armadillo (Arm, β-catenin homolog) plays a key role in transducing the canonical Wnt/ Wg signaling by acting as a transcriptional regulator 4. Arm is also an essential component of adherens junctions (AJs) together with E-cadherins (E-cad) and β-catenin in epithelial cells 5,6. In the absence of Wg signal, Arm is accumulated at the adherens junctions (AJs) where it forms a complex with E-cad or is rapidly degraded 7. Expression of Wg target genes is activated when stabilized Arm enters the nucleus and recruits other co-activators 8. Arm is required for development of fly organs at different stages. It has been shown that loss of Klp64D causes mislocalization of Arm in differentiating photoreceptor cells of pupal retina 9. Klp64D, the Drosophila homolog of Kif3A, is a subunit of the kinesin-II microtubule-based heterotrimeric motor protein complex 10. Recently, we have reported that Klp64D is also required for Wg signaling as a binding partner of Arm 11. Klp64D recruits Arm and its interacting partner Dishevelled (Dsh) for Wg signal transduction. In wing development, Wg signaling is induced from the dorso-ventral (DV) boundary of wing imaginal disc. In the absence of kinesin-II function, Arm is abnormally accumulated with intracellular vesicles in t...