Vitamin D is an important regulator of calcium and phosphorus homeostasis in animals. It can be acquired from the diet or synthesised de novo when skin is exposed to UVb. Vitamin D deficiency can lead to a complex of diseases collectively called metabolic bone disease (MBD). Diurnal lizards without access to UVb are prone to develop vitamin D deficiency, even when dietary vitamin D3 is provided. A trial was conducted to determine whether juvenile nocturnal lizards require access to UVb to prevent vitamin D deficiency. All leopard geckos (Eublepharis macularius) were supplemented with dietary vitamin D3. One group was exposed to low level UVb radiation (33–51 μW/cm2) from hatching until 6 months of age and a second group remained unexposed. Animals were fed ad libitum and their growth and weight gain compared with non-exposed controls. At the end of the trial, blood samples were analysed for vitamin D3 metabolites. The concentration of the vitamin D3 metabolite, 25(OH)D3, was higher in UVb exposed animals (61 ± 20 vs. 38 ± 8 nmol/L), confirming cutaneous synthesis with UVb exposure. Growth and weight gain were similar in both groups, and this, together with the absence of clinical symptoms, suggests that dietary vitamin D3 alone can meet the vitamin D requirements for growth of this nocturnal gecko, during the first six months of life. It remains to be investigated whether the higher vitamin D metabolite levels holds other health benefits for this species, such as improved bone density or immune response.