Steroidal glycoalkaloids (SGAs) are specialized metabolites primarily produced by Solanaceae plants such as potatoes and tomatoes. Notably, α-solanine and α-chaconine are recognized as toxic substances in potatoes. While the biosynthetic pathways of SGAs are largely understood, the final steps of α-solanine and α-chaconine biosynthesis remained elusive. In this study, we discovered that two reductase-encoding genes, reductase for potato glycoalkaloid biosynthesis 1 (RPG1) and RPG2, complete SGA biosynthesis in potato. Knockout of both RPG1 and RPG2 in potato hairy roots halted α-solanine production, leading to the accumulation of zwittersolanine. We analyzed the catalytic function of recombinant enzymes and conducted structural determination of the reaction products by nuclear magnetic resonance. As a result, RPG1 converted zwittersolanine to 16-iminiumsolanine, and RPG2 further converted it to α-solanine. RPG2 also transformed zwittersolanine to 22-iminiumsolanine, which RPG1 then converted to α-solanine. Similar processes were observed for α-chaconine synthesis from zwitterchaconine. Due to differences in enzymatic reaction efficiency, the biosynthetic pathway via 16-iminiumsolanine/16-iminiumchaconine was suggested to be predominant in potato. Our results could pave the way for tailoring SGA structures within Solanum plants, enabling the development of Solanum crop varieties with reduced toxicity or enhanced resistance to diseases and pests.