Ultraviolet B radiation (UVB) is required by many vertebrates to stimulate the photobiochemical synthesis of vitamin D. Vitamin D plays many important roles in the body, including assisting in the absorption of calcium at the level of the intestines. Deficiencies in vitamin D can lead to the development of nutritional disease. Leopard geckos (Eublepharis macularius) are naturally nocturnal to crepuscular; therefore, it is not known whether they benefit from UVB radiation. The purpose of this study was to measure 25-hydroxyvitamin D3 concentrations in leopard geckos exposed to short duration UVB light. Twelve adult, male leopard geckos were used for this study. Blood samples were collected from the cranial vena cava to establish baseline 25-hydroxyvitamin D3 concentrations. Once the baseline samples were collected, the animals were randomly divided into two groups. The animals provided UVB radiation were exposed to non-UVB producing light for 12 h and UVB for 2 h, whereas animals in the control group only received non-UVB producing light for 12 h. Exposure to the UVB light occurred for 2 h per day: 1 h at 0600 h and 1 h at 1800 h to mimic dawn and dusk, respectively. An additional blood sample was collected 30 days after the initiation of UVB exposure. There was a significant difference (F = 9.7, P = 0.012) in 25-hydroxyvitamin D3 concentrations between the two groups, with UVB exposed geckos having significantly higher concentrations. The results of this study demonstrate that short duration exposure to UVB light can lead to increased circulating 25-hydroxyvitamin D3 concentrations in leopard geckos.