Steroidal glycoalkaloids (SGAs) are specialized metabolites in Solanaceae that serve as defensive compounds and undergo significant compositional changes during fruit ripening. This study explored the roles of transcription factors SlNOR and SlNOR-like1 in SGAs biosynthesis during tomato fruit development. UPLC-MS/MS revealed dynamic changes in four major SGAs: tomatidine, β-tomatine, α-tomatine, and Esculeoside A. Transgenic studies with knockout and overexpression lines demonstrated that both SlNOR and SlNOR-like1 positively regulated SGAs accumulation. RT-qPCR analysis showed that these transcription factors modulated multiple GAME genes in the SGAs biosynthetic pathway. Through EMSA and DLR assays, we established that SlNOR and SlNOR-like1 directly bound to and activated GAME25 and GAME40 promoters, two key genes involved in tomatidine synthesis and α-tomatine conversion, respectively. These findings reveal a previously unknown regulatory mechanism of SGAs metabolism and suggest potential strategies for optimizing tomato fruit quality through molecular breeding.