In this study, we investigated the role of macrophage stimulating 1 (Mst1) and the AMPK-Sirt1 signaling pathway in the oxidative stress-induced mitochondrial dysfunction and apoptosis seen in rheumatoid arthritis-related fibroblast-like synoviocytes (RA-FLSs). Mst1 mRNA and protein expression was significantly higher in hydrogen peroxide (HO)-treated RA-FLSs than untreated controls. HO treatment induced the mitochondrial apoptotic pathway by activating caspase3/9 and Bax in the RA-FLSs. Moreover, HO treatment significantly reduced mitochondrial membrane potential and mitochondrial state-3 and state-4 respiration, but increased reactive oxygen species (ROS). Mst1 silencing significantly reduced oxidative stress-induced mitochondrial dysfunction and apoptosis in RA-FLSs. Sirt1 expression was significantly reduced in the HO-treated RA-FLSs, but was higher in the HO-treated Mst1-silenced RA-FLSs. Pretreatment with selisistat (Sirt1-specific inhibitor) or compound C (AMPK antagonist) significantly reduced the viability and mitochondrial function in HO-treated Mst1-silenced RA-FLSs by inhibiting Sirt1 function or Sirt1 expression, respectively. These findings demonstrate that oxidative stress-related upregulation and activation of Mst1 promotes mitochondrial dysfunction and apoptosis in RA-FLSs by inhibiting the AMPK-Sirt1 signaling pathway. This suggests the Mst1-AMPK-Sirt1 axis is a potential target for RA therapy.