SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. This study identifies SIRT6 as an NAD-dependent histone H3K9 deacetylase that regulates telomeric chromatin structure and function. SIRT6 specifically associates with telomeres and its depletion leads to telomere dysfunction, end-to-end chromosomal fusions, and premature cellular senescence. SIRT6-depleted cells exhibit telomere structures similar to those seen in Werner syndrome, a premature aging disorder. SIRT6 deacetylates H3K9 at telomeric chromatin and is required for the stable association of WRN, a protein mutated in Werner syndrome. These findings suggest that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which is essential for proper telomere metabolism and function. The study also shows that SIRT6 is crucial for maintaining a normal replicative lifespan and preventing premature senescence in human cells. SIRT6 knockdown leads to accelerated senescence and telomere dysfunction due to stochastic telomere sequence loss. The premature senescence of SIRT6-depleted cells is attributed to telomere dysfunction rather than defects in base excision repair or oxidative stress. SIRT6 knockdown in U2OS cells results in delayed completion of S phase, consistent with a role for SIRT6 in modulating telomeres during replication. SIRT6 is required for the maintenance of low physiological levels of H3K9 acetylation at telomeric chromatin in S phase. Hyperacetylation of H3K9 in the absence of SIRT6 correlates with telomere dysfunction. SIRT6 is also required for the stabilization of WRN at telomeric chromatin, which is essential for efficient telomere replication and to prevent structural abnormalities at telomeres. These findings suggest that SIRT6 collaborates with WRN at telomeric chromatin to ensure efficient telomere replication and to prevent the accumulation of structural abnormalities at telomeres. The study provides the first identification of a physiological enzymatic activity of SIRT6 and links chromatin regulation by SIRT6 to telomere maintenance and a human premature aging syndrome. SIRT6 is essential for chromatin regulation at mammalian telomeres and provides a new mechanism by which the regulation of chromatin is linked to telomere function, cellular senescence, and potentially organismal aging.SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. This study identifies SIRT6 as an NAD-dependent histone H3K9 deacetylase that regulates telomeric chromatin structure and function. SIRT6 specifically associates with telomeres and its depletion leads to telomere dysfunction, end-to-end chromosomal fusions, and premature cellular senescence. SIRT6-depleted cells exhibit telomere structures similar to those seen in Werner syndrome, a premature aging disorder. SIRT6 deacetylates H3K9 at telomeric chromatin and is required for the stable association of WRN, a protein mutated in Werner syndrome. These findings suggest that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which is essential for proper telomere metabolism and function. The study also shows that SIRT6 is crucial for maintaining a normal replicative lifespan and preventing premature senescence in human cells. SIRT6 knockdown leads to accelerated senescence and telomere dysfunction due to stochastic telomere sequence loss. The premature senescence of SIRT6-depleted cells is attributed to telomere dysfunction rather than defects in base excision repair or oxidative stress. SIRT6 knockdown in U2OS cells results in delayed completion of S phase, consistent with a role for SIRT6 in modulating telomeres during replication. SIRT6 is required for the maintenance of low physiological levels of H3K9 acetylation at telomeric chromatin in S phase. Hyperacetylation of H3K9 in the absence of SIRT6 correlates with telomere dysfunction. SIRT6 is also required for the stabilization of WRN at telomeric chromatin, which is essential for efficient telomere replication and to prevent structural abnormalities at telomeres. These findings suggest that SIRT6 collaborates with WRN at telomeric chromatin to ensure efficient telomere replication and to prevent the accumulation of structural abnormalities at telomeres. The study provides the first identification of a physiological enzymatic activity of SIRT6 and links chromatin regulation by SIRT6 to telomere maintenance and a human premature aging syndrome. SIRT6 is essential for chromatin regulation at mammalian telomeres and provides a new mechanism by which the regulation of chromatin is linked to telomere function, cellular senescence, and potentially organismal aging.