ABSTRACT The study of cellular metabolism has been rigorously revisited over the past decade, especially in the field of cancer research, revealing new insights that expand our understanding of malignancy. Among these insights is the discovery that various metabolic enzymes have surprising activities outside of their established metabolic roles, including in the regulation of gene expression, DNA damage repair, cell cycle progression and apoptosis. Many of these newly identified functions are activated in response to growth factor signaling, nutrient and oxygen availability, and external stress. As such, multifaceted enzymes directly link metabolism to gene transcription and diverse physiological and pathological processes to maintain cell homeostasis. In this Review, we summarize the current understanding of non-canonical functions of multifaceted metabolic enzymes in disease settings, especially cancer, and discuss specific circumstances in which they are employed. We also highlight the important role of subcellular localization in activating these novel functions. Understanding their non-canonical properties should enhance the development of new therapeutic strategies for cancer treatment.