Objective Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. Methods The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence- activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. Results Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. Conclusion These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.