The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa2+) <1.0mM, without recumbency] on physiological responses and function of immune cells in dairy cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43g of oral Ca, as Ca sulfate and Ca chloride, at −1 and 11h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa2+ chelator, intravenous infusion] treatment for 24h, using a crossover design. The sequence of treatments was either NC–SCHI or SCHI–NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa2+ <1.0mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, β-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa2+ in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa2+ concentrations throughout the 24-h treatment period (0.77±0.01 vs. 1.26±0.01mM iCa2+). Induction of SCH reduced dry matter intake on the day of infusion (5.3±0.8 vs. 9.1±0.8kg/d) and rumen contractions (1.9±0.2 vs. 2.7±0.2 contractions/2min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44±0.23 vs. 2.32±0.23ng/mL) evident between 6 and 18h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40±0.04 vs. 4.17±0.04mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110±0.019 vs. 0.061±0.014mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa2+ after stimulation with ionomycin (9.9±1.0 vs. 13.6±1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1±2.1 vs. 29.3±2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1±1.7 vs. 24.2±1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.