The 60 kDa heat shock chaperonin protein 60 (Hsp60 or Cpn60) is highly conserved in evolution and present in nearly all organisms. Eukaryotic paralogues are nuclear encoded but act in certain intra- and extracellular compartments including venous blood. HSP60s function as ATP-dependent molecular chaperones and collaborate with further chaperones to perform their duties. While intracellular HSP60s are crucially involved in proteostasis as well as pro-apoptotic and pro-survival pathways, membrane-bound and extracellular HSP60s are thought to function as danger signals to the immune system and act as powerful immune mediators. HSP60s are released into the peripheral blood from healthy subjects and under a variety of pathological conditions, including cardiovascular diseases and cancer. Hsp60 levels successively rise or decline during tumorigenesis in diverse organs. Hsp60 over-expression in cancer cells stimulates cell proliferation, blocks senescence as well as stress-induced apoptosis, and facilitates oncogenic transformation. This chaperone might thus have future applicability as biomarker for diagnosis and assessing prognosis or response to therapeutic intervention. Hsp60 chaperonopathies represent the basis for targeting Hsp60 for the development of novel agents affecting its activity. This review summarizes recent knowledge and new perspectives on the Hsp60 chaperone machinery and its role in disease and therapy.