Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors are a class of small molecule inhibitors that has led to a profound change in the treatment of breast cancer. The CDK4/6-cyclin D axis mediates cancer cell proliferation, and CDK4/6 inhibitors are known to effectively prevent RB phosphorylation and induce cancer cell cycle arrest. Recent reports have suggested that CDK4/6 inhibition (CDK4/6i) can also have other effects in cancer cells, modulating their immunogenicity, apoptotic responses, and differentiation. Using human breast cancer cell lines and mouse models, we performed chromatin profiling, analysis of 3D conformation, and gene expression analysis to show that CDK4/6 inhibition induces dramatic remodelling of cancer cell chromatin. In addition to promoter inactivation of cell cycle related genes, CDK4/6i causes an unexpected activation of many enhancers and superenhancers at genes involved in apoptotic evasion and tumour cell differentiation. We show that CDK4/6i’s effect on chromatin remodelling is through RB, as RB knockdown prevented the formation of new active enhancers. Importantly, CDK4/6 inhibition increases expression of several Activator Protein-1 factors, which are likely implicated in the activity of these new enhancers. Our findings shed new insights into CDK4/6 pathway biology, and have implications for the future development of CDK4/6 inhibitors.