Gulonolactone oxidase (GLO) is the enzyme responsible for the last step of ascorbic acid biosynthesis. The aim of this study was to investigate the effect of dietary α-tocopherol and ascorbic acid on GLO activity in a lower vertebrate, the white sturgeon (Acipenser transmontanus). Both α-tocopherol and ascorbic acid modulated renal GLO activity. The increase of dietary levels of α-tocopherol and/or ascorbic acid significantly raised the liver concentrations of these two antioxidants and concomitantly lowered kidney’s GLO activity. The results suggest that the enzyme of ascorbic acid synthetic pathway responded to the animal’s antioxidant status and that its activity was downregulated by α-tocopherol. This is the first record of α-tocopherol being involved in the regulation of ascorbic acid synthesis. This new observation may provide a hypothesis for the evolutionary loss of GLO expression in teleost fishes.