The significant rise in the number of diabetic patients worldwide, as well as the development of new insulin delivery techniques such as inhalation or oral administration which require higher dosages, are expected to increase the demand for recombinant insulin. Current manufacturing technologies will be unable to fulfill the rising demand for inexpensive insulin due to their production capacity limitations and high production costs. Production of therapeutic recombinant insulin requires a suitable host organism with adequate post-translational modification and refolding machinery. E. coli and S. cerevisiae have been used extensively to make recombinant human insulin for medicinal applications. However, transgenic plants are particularly appealing expression systems as they can be used to synthesize huge amounts of insulin for human medicinal purposes. Plant-based expression systems have the potential for high-capacity insulin synthesis at a minimal cost. The significant production of biologically active proinsulin in seeds or leaves with long-term stability provides a low-cost technique to develop proinsulin for both injectable and oral administration. Recently, stem cell therapy is being utilized for the treatment of diabetes, as these cells are capable of differentiating into insulin producing c ells. With the advancement of regenerative medicine research for different chronic diseases, treatment for type 1 diabetes mellitus has been reported. The current review concentrates on several biotechnological attributes applied to the rapid and mass synthesis of biologically active insulin and its analogs in microbes, various types of stem cells and transgenic crops.