Steroidal alkaloids (SAs) and their glycosylated forms (SGAs) are toxic compounds largely produced by members of the Solanaceae and Liliaceae plant families. This class of specialized metabolites serves as a chemical barrier against a broad range of pest and pathogens. In humans and animals, SAs are considered anti-nutritional factors because they affect the digestion and absorption of nutrients from food and might even cause poisoning. In spite of the first report on SAs nearly 200 years ago, much of the molecular basis of their biosynthesis and regulation remains unknown. Aspects concerning chemical structures and biological activities of SAs have been reviewed extensively elsewhere; therefore, in this review the latest insights to the elucidation of the SAs biosynthetic pathway are highlighted. Recently, co-expression analysis combined with metabolic profiling revealed metabolic gene clusters in tomato and potato that contain core genes required for production of the prominent SGAs in these two species. Elaborating the knowledge regarding the SAs biosynthetic pathway, the subcellular transport of these molecules, as well as the identification of regulatory and signaling factors associated with SA metabolism will likely advance understanding of chemical defense mechanisms in Solanaceae and Liliaceae plants. It will also provide the means to develop, through classical breeding or genetic engineering, crops with modified levels of anti-nutritional SAs.