Many enzyme-catalyzed reversible reactions can be altered to irreversible processes by proper manipulation of the properties of solvent, substrate and biocatalyst, and by coupling with other irreversible reactions. This review highlights some irreversible strategies applicable to the syntheses of esters, peptides, nucleosides and carbohydrates. Irreversible transesterification can be effected by using enol esters as substrates. Transesterification in aqueous solution can also be realized by antibody catalysis. Either thermodynamic approach or kinetic approach can be employed for irreversible peptide bond formation. The peptide ester with a good ester leaving group is used for the aminolysis reaction catalyzed by subtilisin in aqueous dimethylformamide solution, in which the hydrolysis is minimized. Methylchymotrypsin and other engineered proteases such as subtilisins can also be used to enhance aminolysis over hydrolysis. Modified purine nucleosides such as 7-methylguanosine and 7-methylinosine have been used in the enzymatic transribosylation, wherein the leaving group would undergo an isomerization and render the reaction essentially irreversible. A thermodynamically unfavorable reaction can be driven by coupling with a favorable cofactor regeneration or by using a combined enzyme system. For example, NeuAc aldolasecatalyzed reaction is reversible, but the reaction can be coupled with an irreversible formation of the cytidine 5′-monophosphate CMP-NeuAc. A multiple enzymatic system consisting of glycosyltransferases and kinases has been used to prepare a tetrasaccharide antigen sialyl Lex (NeuAc-Gal-Fuc-GlcNAc) in one pot with regeneration of CMP-NeuAc and formation of NeuAc in situ. A trisaccharide NeuAc-Gal-GlcNAc has been synthesized by the combined reactions with galactosidase and sialyltransferase. 1. Introduction 2. Alteration of Reaction Pathways in Organic Solvents 3. Substrates for Irreversible Transesterification and Transribosylation 4. Solvent Effect in Thermodynamic Peptide Synthesis 5. Solvent and Substrate Effects in Kinetic Peptide Synthesis 6. Modified Biocatalysts in Ester and Peptide Syntheses 7. Irreversible Reactions Assisted with Cofactor Regeneration or Multiple-Enzyme Coupling 8. Conclusion