Feeding a negative dietary cation-anion difference (-DCAD) ration has proven to be an effective method of preventing hypocalcemia. Urine pH is a common method of determining level of diet acidification. Furthermore, urine analyses of Ca, Mg, and P are indicators of mineral status in the cow, and urine deoxypyridinoline (DPD) is a common measurement for bone turnover. Multiparous, nonlactating, non-pregnant Holstein cows were fed -DCAD rations with low, medium, or high concentrations of Ca for 21 d and subjected to a controlled induction of hypocalcemia with 5% ethylene glycol tetraacetic acid (EGTA). Low Ca (LC) cows (n = 5) were fed 0.45% Ca, medium Ca (MC) cows (n = 6) were fed 1.13% Ca, and high Ca (HC) cows (n = 6) were fed 2.02% Ca. Average DCAD was −15.1 mEq/100g DM. Urine and blood samples were collected and urine pH measured daily during the feeding period. Cows were reduced to 60% of preinfusion blood ionized Ca (iCa) during the EGTA challenge, and evaluated until they reached 90% of preinfusion concentrations. A urine sample was collected before the start of EGTA challenge and 30 min later. Urine pH was less than 6.0 in all cows, but was lowest in LC cows during the feeding period (P = 0.02). Blood pH, hematocrit, and serum total Ca, Na, K, total protein and serotonin did not differ among treatments during the feeding period. Blood iCa and glucose were significantly elevated in HC compared with the LC and MC cows during the feeding period (P < 0.05). All urine analyses were corrected to creatinine concentration. Urine Ca, P, Mg and DPD did not differ among treatments at any time. During the EGTA challenge, urine pH decreased over time (P = 0.005), but was not different among treatments. These data suggest that urine mineral concentrations remained relatively constant regardless of dietary calcium, and were unaffected by EGTA challenge. Additionally, a HC diet increased blood iCa and glucose concentrations during the feeding period compared with MC and LC cows, suggesting feeding HC rations with -DCAD may improve metabolic markers of health in the dairy cow.