The evolutionarily conserved WD40 protein budding uninhibited by benzimidazole 3 (BUB3) is known for its function in spindle assembly checkpoint control. In the model plant Arabidopsis thaliana, nearly identical BUB3;1 and BUB3;2 proteins decorated the phragmoplast midline through interaction with the microtubule-associated protein MAP65-3 during cytokinesis. BUB3;1 and BUB3;2 interacted with the carboxy-terminal segment of MAP65-3 (but not MAP65-1), which harbours its microtubule-binding domain for its post-mitotic localization. Reciprocally, BUB3;1 and BUB3;2 also regulated MAP65-3 localization in the phragmoplast by enhancing its microtubule association. In the bub3;1 bub3;2 double mutant, MAP65-3 localization was often dissipated away from the phragmoplast midline and abolished upon treatment of low doses of the cytokinesis inhibitory drug caffeine that were tolerated by the control plant. The phragmoplast microtubule array exhibited uncoordinated expansion pattern in the double mutant cells as the phragmoplast edge reached the parental plasma membrane at different times in different areas. Upon caffeine treatment, phragmoplast expansion was halted as if the microtubule array was frozen. As a result, cytokinesis was abolished due to failed cell plate assembly. Our findings have uncovered a novel function of the plant BUB3 in MAP65-3-dependent microtubule reorganization during cytokinesis.