Actin microfilaments are cytoskeletal structures that constitute the major cytoskeleton in higher plant cells and are often considered to co-localize with microtubule structures. In this study, we observed the interaction of actin microfilaments and microtubules in transgenic tobacco BY-2 cells, dual-visualizing these two cytoskeletal structures using fluorescent protein-labeled cytoskeletal marker proteins. In combination with pharmacological studies, we focused on the role of actin microfilaments in phragmoplast guidance to the proper cortical division zone (CDZ) and discuss their role in increasing the chance to meet the CDZ, to recognize the CDZ, and to guide microtubules to the CDZ.