INTRODUCTION: 5-HMF (5-hydroxymethylfurfural), an active constituent found in Radix Rehmanniae Preparata, a widely utilized traditional Chinese medicine for osteoarthritis (OA) treatment, exhibits notable therapeutic benefits in countering the catabolic and inflammatory responses of OA chondrocytes. Despite these promising effects, the underlying mechanisms of 5-HMF's action remain elusive, thereby impeding its broader clinical application and development. OBJECTIVE: To investigate the impact of 5-HMF on the progression of OA and elucidate its underlying mechanisms. METHODS: In this study, Destabilization of the Medial Meniscus (DMM) was used to construct an OA model of C57BL/6 and transgenic mice in vivo, and interleukin -1β (IL-1β) was used to construct an OA model in vitro. Micro-CT and Alcnohistochemistry (IHC) and immunofluorescence (IF) were used to determine the eian Blue/Hematoxylin and Orange G (ABH/OG) staining were used to observe the morphological changes of joints. Western blot, Polymerase Chain Reaction (PCR), immuxpression levels of cartilage metabolic markers Collagen type II alpha 1 (Col2a1) and Matrix Metalloproteinase-13 (MMP13), as well as glucose transporter Glucose Transporter Type 1 (Glut1), glucose metabolic markers Hexokinase 1 (HK1) and Lactate Dehydrogenase A (LDHA). RNA-seq and Reactom analysis were used to predict the potential mechanism of 5-HMF in the treatment of OA. RESULTS: 5-HMF demonstrates effective alleviation of OA progression, improvement of subchondral sclerosis and cartilage degeneration, particularly in the realm of cartilage protection, which is equivalent to that of celebrex. The protective effect of 5-HMF on cartilage is primarily attributed to its regulatory role in cartilage matrix metabolism, suppress the activity of MMP13 and enhance the expression of Col2a1 to delay cartilage injury. Moreover, RNA sequencing results indicate that 5-HMF's therapeutic effect on OA is closely linked to metabolism, specifically glucose metabolism. Our in vivo and in vitro experiments validate these findings. 5-HMF can counteract the decline in glucose metabolism induced by OA through the Glut1/HK1/LDHA signaling pathway. Furthermore, our findings confirm that Glut1 knockout mice with a DMM-induced OA model do not respond to 5-HMF treatment. CONCLUSION: Our data reveal for the first time that 5-HMF may play a role in cartilage protection in the treatment of osteoarthritis by regulating glycolysis driven by Glut1/HK1/LDHA.