The aim of the present study was to investigate the effects of vascular tissue levels of l‐carnitine and its precursor, γ‐butyrobetaine (GBB), on the development of endothelial dysfunction induced by 5 μmol/L lysophosphatidylcholine (LPC), 10 mmol/L triglycerides (TG) or a high glucose concentration (44 mmol/L). Changes in vascular tissue levels of l‐carnitine and GBB were induced by administration of l‐carnitine (100 mg/kg), mildronate (100 mg/kg; an inhibitor of l‐carnitine synthesis) or their combination to male Wistar rats for 2 weeks. Treatment with l‐carnitine elevated vascular tissue levels of l‐carnitine, whereas administration of mildronate reduced l‐carnitine levels and increased GBB levels. Experimental animals that received the combination of both drugs showed elevated tissue levels of GBB. The results from organ bath experiments demonstrated that increased GBB levels with preserved l‐carnitine content in vascular tissues attenuated the development of endothelial dysfunction induced by high glucose. However, changes in vascular tissue l‐carnitine and GBB levels had no impact on endothelial dysfunction induced by TG or LPC. The results demonstrate that increased levels of GBB with preserved l‐carnitine content in vascular tissue attenuate the development of endothelial dysfunction induced by high glucose concentrations.