The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.