Atherosclerotic cardiovascular disease is accompanied by changes in arterial connective tissue. We evaluated the effects of fat-soluble vitamins A, D, and E individually and in combinations on the composition of extracellular matrix produced and deposited by arterial wall cells, human aortic smooth muscle cells, and endothelial cells. Individually, vitamins D and E stimulated collagen type I extracellular matrix deposition in human aortic smooth muscle cell cultures. However, vitamins A, D, and E reduced collagen type IV deposition by human aortic smooth muscle cell, counteracting the stimulatory effects of vitamin C. The extracellular matrix deposition of heparan sulfate by human aortic smooth muscle cells increased by vitamin C and its combination (C+D+E). β-carotene + D + C induced the extracellular matrix deposition of collagen I by endothelial cells. Vitamin E with other vitamins resulted in either induction (E+C+A) or inhibition (E+D). The extracellular matrix deposition of type IV collagen and elastin by human aortic endothelial cells was not affected by test vitamins, except the extracellular matrix type IV collagen decrease by combinations (A+E), (A+D+E), and (C+D+E). The extracellular matrix deposition of all tested glycosaminoglycans was reduced by vitamin A and its combination (A+C+D+E). Therefore, the fat-soluble vitamins applied individually or in combination—both with each other or with ascorbic acid—can affect extracellular matrix deposition of type I and IV collagens, and key glycosaminoglycans by cultured human aortic arterial wall cells.