For several decades, dermatologists have utilized azathioprine to treat numerous debilitating skin diseases. This synthetic purine analog is derived from 6-mercaptopurine. It is thought to act by disrupting nucleic acid synthesis and has recently been found to interfere with T-cell activation. The most recognized uses of azathioprine in dermatology are for immunobullous diseases, generalized eczematous disorders, and photodermatoses. In this comprehensive review, the authors present recent advancements in the understanding of azathioprine and address aspects not covered in prior reviews. They (1) summarize the history of azathioprine; (2) discuss metabolism, integrating information from recent publications; (3) review the mechanism of action with attention paid to the activities of azathioprine not mediated by its 6-mercaptopurine metabolites and review new data about inhibition by azathioprine of the CD28 signal transduction pathway; (4) thoroughly examine thiopurine s-methyltransferase genetics, its clinical relevance, and interethnic variations; (5) review prior uses of azathioprine in the field of dermatology and grade the level of evidence; (6) discuss the use of azathioprine in pregnancy and pediatrics; review (7) key drug interactions and (8) adverse effects; (9) suggest a dosing and monitoring approach different from prior recommendations; and (10) explore the future of azathioprine, focusing on laboratory considerations and therapeutic application.