Prepartum feeding of a negative dietary cation-anion difference (−DCAD) ration has proved to be an effective method of preventing hypocalcemia. However, the optimum Ca concentration to feed with −DCAD rations has not been determined. We conducted an experiment in multiparous nonlactating, non-pregnant Holstein cows fed −DCAD rations (15.1 mEq/100g DM) for 21 d with low (LC;0.45% Ca;n = 5), medium (MC;1.13% Ca;n = 6) or high (HC;2.02% Ca;n = 6) concentrations of dietary Ca and then subjected to a controlled induction of hypocalcemia to determine the ability of cows to respond to the challenge based on dietary Ca. On d 22, 23, and 24 hypocalcemia was induced using an intravenous infusion of 5% ethylene glycol tetraacetic acid (EGTA). During infusion, blood samples were collected every 15 min until 60% of preinfusion iCa concentrations were achieved. Samples were collected post-infusion at 0, 2.5, 5, 10, 15, 30, and every 30 min thereafter until 90% of preinfusion iCa was reached. HC cows maintained higher concentrations of iCa during infusion period and had a longer average time of EGTA infusion to 60% iCa (414.2 ± 38.9min vs. 242 ± 28.0min MC and 282 ± 24.8min LC; P = 0.03). More EGTA was required to reach 60% iCa in HC cows (16.6 ± 1.5g vs. 10.3 ± 1.3 g in MC and 12.3 ± 1.2 g in LC; P = 0.01). Using a regression analysis and forced linearity to compare slopes of lines during EGTA challenge, MC cows reached 60% iCa faster than HC and LC cows in the first 145 min of infusion (P < 0.0001). Total serum Ca decreased in all cows during infusion but not differ between treatment (P = 0.59). Total serum protein tended to be elevated in HC cows compared with MC and LC cows during EGTA challenge (P = 0.10). Serotonin concentrations were elevated in MC cows (7,076 ± 914.5 ng/mL) compared with HC (5,530.39 ± 806.1ng/mL) and LC (3,621.34 ± 890.9 ng/mL) cows during EGTA challenge (P = 0.05), suggesting an interdependent relationship between iCa and serotonin. Cows fed HC had increased resistance to induction of hypocalcemia as measured by iCa, indicating potential metabolic benefits of feeding a higher Ca diet when using a −DCAD ration.