Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the non-histone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV(12), and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly.