Inactivation of the retinoblastoma (RB) tumor suppressor pathway, via elevated cyclin-dependent kinase (CDK) activity, is observed in majority of human cancers. Since CDK deregulation is evident in most cancer cells, pharmacological CDK inhibition has become an attractive therapeutic strategy in oncology. We recently showed that an oncogenic CDK4(R24C) mutation alters the subcellular localization of the normally nuclear RB phosphoprotein. Here, using 71 human cancer cell lines and over 300 primary human cancer tissues, we investigated whether changes in RB subcellular localization occur during human cancer progression. We uncover that diverse human cancers and their derived cell lines, particularly those with poor tumor differentiation, display significant cytoplasmic mislocalization of ordinarily nuclear RB. The nucleocytoplasmically distributed RB was derived via CDK-dependent and Exportin1-mediated nuclear export. Indeed, cytoplasmically mislocalized RB could be efficiently confined to the nucleus by pharmacologically reducing CDK activity or by inhibiting the Exportin1-mediated nuclear export pathway. Our observations uncover a post-translational CDK-dependent mechanism of RB inactivation and suggest that cytoplasmically localized RB may harbor a tumor promoting function. We propose that RB inactivation, via aberrant nucleocytoplasmic transport, may disrupt normal cell differentiation programs and accelerate the cancer process. These results are evidence that tumor cells modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.