The tobacco protein kinase NPK1 is a MAPKKK that regulates formation of the cell plate during cytokinesis. In the present study, we have identified tobacco NQK1/NtMEK1 and NRK1 as a MAPKK and a MAPK, respectively, downstream of NPK1. NQK1/NtMEK1 complements the mutation in the PBS2 MAPKK gene of yeast in a manner that depends on both NPK1 and its activator, NACK1, a kinesin-like protein. Active NPK1 and NQK1/NtMEK1 phosphorylate and activate NQK1/NtMEK1 and NRK1, respectively. Both NQK1/NtMEK1 and NRK1, as well as NPK1, are activated at the late M phase of the cell cycle in tobacco cells, and they are rapidly inactivated by depolymerization of phragmoplast microtubules. These results suggest the existence of a MAPK cascade that consists of NPK1, NQK1/NtMEK1, and NRK1 and functions in a process related to the architecture of phragmoplasts at the late M phase of the cell cycle. Overexpression of kinase-negative NQK1/NtMEK1 in tobacco cells generates multinucleate cells with incomplete cross-walls. Arabidopsis plants with a mutation in the ANQ1 gene, an ortholog of NQK1/NtMEK1, display a dwarf phenotype, with unusually large cells that contain multiple nuclei and cell-wall stubs in various organs. In addition, anq1 homozygotes set fewer flowers and produce large and malformed pollen grains with a tetrad structure. Thus, NQK1/NtMEK1 (ANQ1) MAPKK appears to be a positive regulator of plant cytokinesis during meiosis as well as mitosis.