Vitamin D is an important hormone in vertebrates, and most animals acquire this hormone through their diet and/or exposure to ultraviolet B (UVB) radiation. To date, no study has determined how chinchillas (Chinchilla laniger) acquire vitamin D. The objective of this study was to determine whether exposure to UVB radiation had an effect on serum 25-hydroxyvitamin D levels in captive chinchillas. Overall, 10 juvenile chinchillas were used for this scientific investigation. Baseline blood samples were collected from the animals while under isoflurane anesthesia to determine their serum 25-hydroxyvitamin D concentrations. The chinchillas were then randomly assigned to 2 treatment groups: Group A, 12 hours of UVB exposure per day and Group B, no UVB exposure. At the end of the study (Day 16), a second blood sample was collected from each animal while again under isoflurane anesthesia to measure serum 25-hydroxyvitamin D concentrations. Mean ± standard deviation serum 25-hydroxyvitamin D concentrations differed significantly (P = 0.048) between juvenile chinchillas provided supplemental UVB radiation (189.0 ± 102.7 nmol/L) and those not provided supplemental UVB radiation (87.8 ± 34.4 nmol/L). This study found that exposing juvenile chinchillas to UVB radiation significantly increased their circulating serum 25-hydroxyvitamin D levels. Because vitamin D is an essential hormone in vertebrates, these findings suggest that the provision of UVB radiation to captive chinchillas may be important. Further research to elucidate the importance of 25-hydroxyvitamin D and UVB radiation in captive chinchillas is required.