Phosphorus plays a fundamental role in cell since it is a part of many biomolecules and biological metabolites which include phospholipids, nucleic acids, proteins, polysaccharides or nucleotide cofactors. Phosphorus is most commonly found under its highest oxidized state as in orthophosphate or phosphate esters. However, there are also examples of naturally occurring molecules bearing phosphorus atoms at lower oxidized state that contain one carbon to phosphorus (P-C) bond. These compounds, so-called phosphonates, are much more resistant to chemical and enzymatic hydrolysis, thermal decomposition and photolysis than phosphates. The first example of a natural phosphonate, 2-aminoethylphosphonic acid (2-AEP), was reported in 1959 and shown to be a constituent of lipids, proteins and polysaccharides. Since the beginning of the 20th century, with the discovery of the Arbuzov reaction, synthetic phosphonates have been developed which have applications as therapeutic agents (e.g. antibiotics, anti-viral, or trypanocidal drugs) as well as insecticides and herbicides. Recently, phosphonate derivatives have been extensively used as enzyme inhibitors due to their specific structural and electronic properties. First, since they differ from phosphates by the single substitution of one oxygen atom with a carbon atom, phosphonates have been widely used as isosteric mimics of phosphates in the design of analogues of enzyme substrates or cofactors. They offer the advantage of a much greater stability to hydrolysis and resistance to proteases than phosphates and therefore can be used to mimic highly chemically unstable phosphates like carboxy-phosphates. Such a strategy was shown successful for generating competitive inhibitors of glycolytic enzymes (triose/hexose phosphate analogues), phosphatases or viral DNA polymerases (phosphonate nucleotides), with applications as chemotherapeutic agents against trypanosomiasis, hepatitis B or human immunodeficiency virus. Moreover, introduction of one or two fluorine atoms on the α-methylene group was also used to provide a better mimic of phosphate geometric and electronic properties by lowering the pKa of the phosphonate and increasing its P-Cα-Cβ dihedral angle. Phosphonic acids have also been extensively studied as enzyme inhibitors mimicking tetrahedral transition states. They proved to be potent competitive inhibitors of peptidases as mimics of the tetrahedral gem-diolate transition state of peptide bond hydrolysis, and of lipases, as mimics of configuration and charge distribution of the first transition state in triglyceride hydrolysis. This chapter summarizes the most recent studies on phosphonate-based enzyme inhibitors with special emphasis on isosteric analogues of phosphorylated substrates and mimics of tetrahedral transition states for peptide bond hydrolysis.