A colony of guinea pigs (n = 9) with α-mannosidosis was fed a pelleted commercial laboratory guinea pig diet. Over 2 mo, all 9 guinea pigs unexpectedly showed anorexia and weight loss (11.7% to 30.0% of baseline weight), and 3 animals demonstrated transient polyuria and polydipsia. Blood chemistry panels in these 3 guinea pigs revealed high-normal total calcium, high-normal phosphate, and high ALP. Urine specific gravity was dilute (1.003, 1.009, 1.013) in the 3 animals tested. Postmortem examination of 7 animals that were euthanized after failing to respond to supportive care revealed renal interstitial fibrosis with tubular mineralization, soft tissue mineralization in multiple organs, hepatic lipidosis, and pneumonia. Analysis of the pelleted diet revealed that it had been formulated with a vitamin D3 content of more than 150 times the normal concentration. Ionized calcium and 25-hydroxyvitamin D values were both high in serum saved from 2 euthanized animals, confirming the diagnosis of hypervitaminosis D. This report discusses the clinical signs, blood chemistry results, and gross and histologic findings of hypervitaminosis D in a colony of guinea pigs. When unexpected signs occur colony-wide, dietary differentials should be investigated at an early time point.