Par1b is an evolutionarily conserved kinase that plays crucial roles in cell polarity. Controlling intracellular localization of Par1b is important for its biological activity. We previously reported that Wnt stimulation or expression of Dvl promotes accumulation of Par1b in the membrane (T. Terabayashi, T.J. Itoh, H. Yamaguchi, Y. Yoshimura, Y. Funato, S. Ohno, H. Miki, Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons, J. Neurosci. 27 (2007) 13098-13107). However, its molecular mechanism remains unclear. Here we show the importance of Par1b phosphorylation in the regulation of membrane localization. We find that Thr-324 is phosphorylated in a Dvl-dependent manner. Interestingly, the conversion of Thr-324 to Glu results in a significant accumulation of Par1b in the membrane, without any effects on the kinase activity. Moreover, the phospho-mimicking Par1b mutant does not antagonistically function against Dvl in microtubule stabilization and neurite extension, although wildtype Par1b does. These results suggest that membrane accumulation of Par1b induced by Dvl is regulated by its phosphorylation status, which is important for Par1b to regulate the microtubule dynamics.