Osteoarthritis (OA) is a common joint disease lacking effective treatments. Dexamethasone (Dex) is often used to relieve joint pain. However, the adverse effects of Dex on cartilage can't be ignored. This study aimed to investigate the effect of Dex on articular cartilage and its mechanism by in vitro and in vivo experiments. The results showed that intra-articular injection with Dex damaged the matrix synthesis of cartilage. In vitro, Dex induced human chondrocytes mitochondrial dysfunction and increased reactive oxygen species (ROS) level, while down-regulated or unchanged key glycolysis genes, but increased lactic acid (LA) concentration. It was showed that high concentrations of LA induced chondrocytes apoptosis. Mechanistically, monocarboxylate transporter 4 (MCT4) was inhibited by Dex and had a significant negative correlation with ROS level. Further results showed that the trimethyl-histone H3-K4 (H3K4me3) level of MCT4 was reduced by Dex, and the ROS scavenger N-Acetyl-L-cysteine (NAC) and α-ketoglutarate (α-KG) alleviated the Dex-induced obstruction of matrix synthesis and high level of ROS by up-regulating the H3K4me3 level of MCT4 and its expression. In conclusion, Dex exhibited harm to cartilage, shown as mitochondrial dysfunction and increased ROS. The latter further caused LA accumulation in chondrocytes via decreasing the H3K4me3 level of MCT4 and its expression, which may account for the long-term side effects of Dex on chondrocytes. And α-KG may be used as an auxiliary drug to weaken the toxic effect of Dex on cartilage.