The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. Here, we report that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. We describe a novel interaction between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability.