Primary human cells enter senescence after a characteristic number of population doublings (PDs). In the current study, human skin fibroblasts were propagated in culture under 5.5 mM glucose (normoglycemia); addition of 16.5 mM d-glucose to a concentration of 22 mM (hyperglycemia); and addition of 16.5 mM l-glucose (osmotic control). Hyperglycemia induced premature replicative senescence after 44.42±1.5 PDs compared to 57.9±3.83 PDs under normoglycemia ( p<0.0001). l-Glucose had no effect, suggesting that the effect of hyperglycemia was not attributed to hyperosmolarity. Activated caspase-3 measurement showed a significantly higher percentage of apoptotic cells in high glucose medium. Telomerase overexpression circumvented the effects of hyperglycemia on replicative capacity and apoptosis. The “point of no return,” beyond which hyperglycemia resulted in irreversible progression to premature replicative senescence, occurred after exposure to hyperglycemia for as few as 20 PDs. These results may provide a biochemical basis for the relationship between hyperglycemia and those complications of diabetes, which are reminiscent of accelerated senescence.