Silk is a natural engineering material with a unique set of properties. The major constituent of silk is fibroin, a protein widely used in the biomedical field because of its mechanical strength, toughness and elasticity, as well as its biocompatibility and biodegradability. The domestication of silkworms allows large amounts of fibroin to be extracted inexpensively from silk cocoons. However, the industrial extraction process has drawbacks in terms of sustainability and the quality of the final medical product. The heterologous production of fibroin using recombinant DNA technology is a promising approach to address these issues, but the production of such recombinant proteins is challenging and further optimization is required due to the large size and repetitive structure of fibroin's DNA and amino acid sequence. In this review, we describe the structure–function relationship of fibroin, the current extraction process, and some insights into the sustainability of silk production for biomedical applications. We focus on recent advances in molecular biotechnology underpinning the production of recombinant fibroin, working toward a standardized, successful and sustainable process.