Enzymesare key biological macromolecules that support life by accelerating theconversion of target molecules to desired products in many biochemical reactions. Enzymes are characterized by high affinity, specificity and great catalytic efficiency. Owing to their unique characteristics, enzymes have attracted significant attention for use in therapeutic settings as a distinct class of drugs different from other types of medicines. Enzyme-based therapies are currently in use for the treatmentof a wide range of diseases, including leukemia, metabolic disorders, inflammation and cardiovascular disease. However, several challenges, such as immunogenicity and stability, remain. X-ray crystallography has provided key structural insights into the understanding of the molecularbasis of diseases and development of enzyme-based therapies. Here, the  role of X-ray crystallography in the development of therapeutic enzymes is examined and several examples are provided.