Insulin therapy remains the most effective method to treat diabetes mellitus (DM), and the demand for this valuable hormone has exceeded that of any other protein-based medicine as a result of the dramatic increase in the number of diabetic patients worldwide. Understanding the structure of insulin and the interaction with its receptor is important for developing proper formulations. As a result of the relatively low thermal stability of native insulin and its two-chain analogues, the application of single-chain insulin (SCI) analogues, which can be obtained relatively easily by recombinant DNA technology or chemical synthetic methods, represents a promising alternative approach. In this review, the basic knowledge of insulin (discovery, biosynthesis, and structure) and the current model of the interaction with its receptor are outlined. Furthermore, we outline the strategies for the design and production of various SCI analogues and their reported applications.