Abstract: Glycolysis serves as the primary metabolic pathway for glucose in endothelial cells (ECs), providing energy and essential substances for EC life activities and also participating in tissue and organ development as well as various pathological processes. Recent studies have revealed that glycolytic enzymes possess the characteristics of "moonlighting proteins" beyond their metabolic role, enabling them to contribute to the maintenance of mitochondrial membrane stability, cell cycle regulation, gene transcription and signal transduction through non-metabolically dependent pathways. Therefore, the moonlighting function of glycolytic enzymes has emerged as a significant novel mechanism for co-regulating EC metabolism and molecular expression. In this review, we comprehensively summarize the regulatory effects and mechanisms underlying the non-metabolic functions of glycolytic enzymes on EC’s biological behavior in both physiological and pathological environments, with an aim to further elucidate the regulatory mechanisms governing EC function and identify new potential therapeutic targets for related vascular diseases.