BackgroundThere are abnormalities in vitamin D metabolism in people with calcium nephrolithiasis, but limited data are available on vitamin D status in dogs with calcium oxalate (CaOx) urolithiasis.ObjectiveTo compare serum concentrations of vitamin D metabolites in dogs with and without hypercalciuric CaOx urolithiasis.AnimalsThirty‐eight dogs with (n = 19) and without (n = 19) a history of CaOx urolithiasis and hypercalciuria.MethodsRetrospective cross‐sectional study. Serum 25‐hydroxyvitamin D [25(OH)D], 1,25‐dihydroxyvitamin D [1,25(OH)2D], and 24,25‐dihydroxyvitamin D [24,25(OH)2D] were measured. The ratios of 25(OH)D/24,25(OH)2D and 1,25(OH)2D/25(OH)D were compared between cases and controls.ResultsThere were no significant differences between cases and controls when comparing 25(OH)D, 24,25(OH)2D, 1,25(OH)2D, or 1,25(OH)2D/25(OH)D. Cases had higher 25(OH)D/24,25(OH)2D (median = 1.40, range = 0.98‐1.58) compared to controls (median = 1.16, range = 0.92‐2.75; P = .01). There was overlap in the ranges for 25(OH)D/24,25(OH)2D between cases and controls, but 6 cases (32%) had ratios above the control dog range. There was a moderate positive correlation between the ratio of 25(OH)D/24,25(OH)2D and urinary calcium‐to‐creatinine ratios (r = 0.40, 95% confidence interval = 0.10‐0.64; P = .01).Conclusions and Clinical ImportanceThese data suggest that decreased conversion of 25(OH)D to 24,25(OH)2D occurs in a subset of dogs with CaOx urolithiasis. Abnormalities in vitamin D metabolism might contribute to stone risk in dogs.