In bone and teeth formation, coordinated calcification is a highly desirable biological process. However, heterotopic calcification at unwanted tissue sites leads to dysfunction, disease and, potentially, to death and therefore requires prevention and treatment. With the recent discovery of calcification inhibitors we now know that biological calcification is not passive but a complex, active and highly regulated process. Calcification at vascular sites is the most threatening localization and manifests as part of atherosclerosis or arteriosclerosis. Atherosclerosis is often accompanied by intimal plaque calcification, whereas arteriosclerosis is characterized by calcification of the media. The severity of calcification of cerebral or coronary atherosclerotic plaques is associated with an increased incidence of events such as stroke or myocardial infarction. Medial calcification is the major cause of arterial stiffness, which contributes to left ventricular dysfunction and heart failure. Patients with chronic kidney disease are at especially increased risk for both intimal and medial calcification. In this context, it is currently thought that calcium-regulatory factors including fetuin-A, matrix Gla protein, osteoprotegerin, and pyrophosphates act in a local or systemic manner to prevent calcifications of the vasculature, and that dysregulations of such calcification inhibitors may contribute to progressive calcifications. Nephrolithiasis represents another process of unwanted calcification responsible for significant morbidity. More than 80% of renal stones contain calcium. Urinary factors inhibiting calcification are citrate, glycosaminoglycans, Tamm-Horsfall protein, and osteopontin. This review summarizes current experimental and clinical data underlining the biological importance of these calcification inhibitors.