Background: Humans take vitamin D supplements to reduce risk of vitamin D deficiency and reduce the risk of osteoporosis. However, it is unclear how dietary super-dose (10,000x greater than requirement) can affect vitamin D status in aged animals. Aged laying hens could potentially be a model to compare with women in peri- or postmenopausal stages of life because their bone health is physiologically taxed from egg production and they are highly susceptible to osteoporosis. Objective: We investigated dietary super-dose impacts of cholecalciferol (vitamin D3) on vitamin D status in aged laying hens in production. Methods: Forty-eight 68-wk old Hy-Line Brown laying hens were individually housed in cages with eight hens per dietary treatment for eleven weeks. Hens were randomly assigned to one of six groups of dietary vitamin D3 supplementation and fed ad libitum. Supplementation levels were 400 (recommended dosage for hens), 800, 7,400, 14,000, 20,000, and 36,000 IU D3/kg of feed. At termination of the study, all hens were euthanized and we collected blood, feces, and tibia and humerus bones. Ionized (free) blood calcium, fecal calcium, bone calcium, and plasma vitamin D metabolites were measured. Results: We did not discern any dietary effects in tissue and fecal calcium. We observed that increasing dietary vitamin D3 increased plasma vitamin D3, 25-hydroxycholecalciferol, and 24,25-dihydroxycholecalciferol concentrations (p < 0.0001 for all 3 metabolites). We also observed super-dose fed hens had decreased kidney 24-hydroxylase expression (p = 0.0006). Conclusions: Although dietary vitamin D3 super-doses did not affect calcium status in our aged laying hens, it is possible there is an age-related effect of not being as sensitive to vitamin D efficacy. We suggest future research should explore how 24-hydroxylation mechanisms are affected by vitamin D supplementation. Further understanding of 24-hydroxylation can help ascertain ways to reduce risk of vitamin D toxicity.