The effects of systemic administration of glucocorticoids (GC) on Achilles tendons of rats was studied. After the animal euthanasia, Achilles tendons were removed in sterile conditions from Sprague Dawley adult female rats to isolate tenocytes. Animals have been daily treated for 8 weeks with 4 mg/kg methylprednisolone or Sham-treated with saline solution. In vitro, cell proliferation (WST-1), extra-cellular matrix (ECM) synthesis (collagen type I, CICP; proteoglycans, PG; and fibronectin, FBN), and transforming growth factor (TGF-β1) were evaluated at 3 and 7 days of culture. The effect of glucocorticoids (GC) on tenocytes was evident both at 3 and 7 days of culture, and caused a significant decreases in cell proliferation (P<0.01), CICP (P<0.01) and PG synthesis (P<0.01) as compared to NT tenocytes. In conclusion, GC systemic treatment seems to compromise the normal proliferation rate and synthetic activity of cultured tenocytes. This study was helpful in understanding the fundamental biological processes that occur during corticosteroid systemic administration and tenocyte cultures may be used to further study to improve knowledge of these cell behaviour also in the ambit of tendon tissue-engineered therapies.