Objectives Vitamin D deficiency accompanies chronic cholestatic liver disease (CLD) in humans. The vitamin D status of cats with CLD is unknown. The objectives of this study were to describe serum vitamin D concentrations in cats with CLD and to determine if they correlated with indices of liver disease severity. Methods Thirty-six cats with CLD, defined by increases in serum bilirubin and serum alanine aminotransferase, and 23 sick cats with non-hepatobiliary diseases were prospectively enrolled. Serum 25-hydroxyvitamin D (25[OH]D), parathyroid hormone (PTH) and ionized calcium were measured. Signalment, clinical signs, comorbidities, diet history, serum bilirubin, liver enzyme activity, albumin, phosphorus, white blood cell count, prothrombin time and final hepatic cytologic/histopathologic diagnosis, when available, were recorded. Results Median serum 25(OH)D levels were similar in cats with CLD (89.5 nmol/l; range 21–112 nmol/l) and sick cats (89.0 nmol/l; range 49–115 nmol/l). Overall 12/36 (33%) of cats with CLD and 4/23 (17%) sick cats had 25(OH)D levels below the lower limit of the reference interval (<65 nmol/l). Median PTH concentrations in cats with CLD were significantly higher (0.95 pmol/l; range 0–11.3 pmol/l) than in sick cats (0.70 pmol/l; range 0.5–6 pmol/l). In cats with CLD, 6/36 (17%) had high PTH levels in contrast to only 1/23 (4%) sick cats. In cats with CLD, 25(OH)D concentrations did not correlate with serum bilirubin, albumin or serum liver enzymes but were moderately negatively correlated with white blood cell count (r = − 0.402, P = 0.013). Cats with hepatic lipidosis had the highest prevalence of 25(OH)D concentrations that fell below the reference interval. Conclusions and relevance Many cats with CLD have serum 25(OH)D concentrations below the lower limit of the reference interval. Further study is warranted to determine the clinical relevance and whether supplementation would provide benefits.