Cytokinesis is the final step in cell division and is driven by the constriction of the medial actomyosin-based contractile ring (CR) in many eukaryotic cells. In the fission yeast Schizosaccharomyces pombe, the IQGAP-like protein Rng2 is required for assembly and constriction of the CR, and specifically interacts with actin filaments (F-actin) in the CR after anaphase. However, the mechanism that timely activates Rng2 has not yet been elucidated. We herein tested the hypothesis that the cytokinetic function of Rng2 is regulated by phosphorylation by examining phenotypes of a series of non-phosphorylatable and phosphomimetic rng2 mutant strains. In phosphomimetic mutant cells, F-actin in the CR was unstable. Genetic analyses indicated that phosphorylated Rng2 was involved in CR assembly in cooperation with myosin-II, whereas the phosphomimetic mutation attenuated the localization of Rng2 to CR F-actin. The present results suggest that Rng2 is phosphorylated during CR assembly and then dephosphorylated, which enhances the interaction between Rng2 and CR F-actin to stabilize the ring, thereby ensuring secure cytokinesis.