Objective The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression. Methods Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNFα-treated extracts was identified by mass spectroscopy, and its amino-terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα. Results TNFα-treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full-length 110-kd protein and a smaller 75-kd fragment. The 75-kd SirT1 fragment was found to lack the carboxy-terminus. Cathepsin B was identified as the TNFα-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75-kd SirT1 fragment. Conclusion These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B-mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage-specific gene expression evident in these TNFα-treated cells.