Protein phosphorylation plays important roles in a variety of stress responses. Although plasmodium of Physarum polycephalum rapidly grows and shows an active cytoplasmic streaming under nutrient and wet conditions, dry stress transforms plasmodium into a dormant state called sclerotium. Sclerotium can change into plasmodium within several hours after addition of water. We herein report that more than half of actin in sclerotium was in a phosphorylated state. The in vivo phosphorylation site was identified to be Thr-203 which is in contact with another actin molecule upon polymerization. The phosphorylated from of actin showed no polymerizing activity, while the unphosphorylated form possessed the ability to polymerize into F-actin. These results suggest that phosphorylation of Physarum actin is involved in reorganization and/or preservation of the actin molecules in the process of sclerotium formation.