The first genes composing the Polycomb group (PcG) were identified 50 years ago in Drosophila melanogaster as essential developmental functions that regulate the correct segmental expression of homeotic selector genes. In the past two decades, what was initially described as a large family of chromatin-associated proteins involved in the maintenance of transcriptional repression to maintain cellular memory of homeotic genes turned out to be a highly conserved and sophisticated network of epigenetic regulators that play key roles in multiple aspects of cell physiology and identity, including regulation of all developmental genes, cell differentiation, stem and somatic cell reprogramming and response to environmental stimuli. These myriad phenotypes further spread interest for the contribution that PcG proteins revealed in the pathogenesis and progression of cancer and other complex diseases. Recent novel insights have increasingly clarified the molecular regulatory mechanisms at the basis of PcG-mediated epigenetic silencing and opened new visions about PcG functions in cells. In this review, we focus on the multiple modes of action of the PcG complexes and describe their biological roles.