It is a hypothesis that autoimmune factors directed against endothelial cells play a role in developing venous insufficiency. We investigated the association between anti-endothelial cell antibodies (AECA) and the development of venous insufficiency and varicose veins. Patients, methods: 44 patients were evaluated with clinical examination and duplex ultrasound for diagnosing chronic venous insufficiency and varicose veins and 120 healthy volunteers were assigned as the control group without evidence of chronic venous insufficiency and varicose veins. All sera samples were analysed by using slides, each containing biochips coated with frozen sections of HUVEC (human umblical vein endothelial cells) and capillary-rich tissue such as skeletal muscle (Euroimmun, FB 1960-1005-2, Germany). If a positive reaction is obtained, specific antibodies of class IgA, IgG, IgM attach to the antigens. In a second step, the bound antibodies are stained with fluorescein labeled antihuman antibodies and visualised by fluorescence microscopy. Results: AECA was positive in 24 out of 44 patients (54.54%) and in 30 out of 120 volunteers (25%). We detected that anti HUVEC antibody occurred significantly more frequent in patients with chronic venous insufficiency or varicose veins: p = 0.0007, OR: 3.60 (1.65 < 7.92). Discussion: The presence of antibodies directed against the endothelial structure causes inflammatory cells of the immune system to move towards the location by both forming antigen-antibody complex and activating the complement system. Tissue damage may occur due to inflammation. In our study we have found a statistically significant relationship between antiendothelial cell antibodies and chronic venous insufficiency. Conclusion: Early diagnosis or prediction of venous insufficiency and/or varicose veins before the occurrence of symptoms may prevent the damage or even help to establish a prophylactic treatment.