Phosphorylation of myosin II is thought to play an important role in cytokinesis. Although it is well known that phosphorylated regulatory light chain of myosin II (P-MRLC) localizes along the contractile ring, it is not clear how P-MRLC controls myosin II and F-actin in furrow ingression during cytokinesis. To elucidate roles of P-MRLC in furrow ingression, HeLa cells transfected with EGFP-tagged wild-type or each MRLC mutant were observed using a live-imaging microscope. Time-lapse observation revealed that a delay of furrow ingression was observed in the nonphosphorylatable form of MRLC (AA-MRLC)-expressing cell but not in the wild-type or phospho-mimic MRLC-expressing cell. Among each form of MRLC-expressing cell, the total amount of P-MRLC including phospho-mimic MRLCs was smallest in the cell expressing AA-MRLC. However, the amount of F-actin and myosin II at the contractile ring in the AA-MRLC-expressing cell was the same as that in the normal cell. Interestingly, delay of furrow ingression by a Rho-kinase inhibitor, Y27632, was rescued by phospho-mimic MRLCs. These results suggest that the P-MRLC is essential for the progress of furrow ingression but not the retainment of F-actin and myosin II in the contractile ring of dividing HeLa cells.