Our objectives were to assess the effects of a negative dietary cation-anion difference (DCAD) on phagocytosis (P) and oxidative burst (OB) function of circulating neutrophils and to determine the associations of serum ionized (iCa) and total calcium concentrations (tCa) with P and OB. We hypothesized that multiparous cows fed a negative DCAD prepartum would have greater serum ionized (iCa) and total calcium concentrations (tCa), and thus improved P and OB. From 3 wk before expected parturition until calving, 38 healthy multiparous cows from 3 farms were randomly assigned to negative DCAD (NEG; −100 meEq/kg DM; n = 21) or a control diet (CON; 12 meEq/kg DM; n = 17). Urine pH was measured weekly and in NEG was 6.3 ± 0.8 with 76% of 21 samples <7 and 62% < 6.5. Each farm was on 1 treatment or the other. P, OB, iCa, and tCa were measured at −7, 1, and 4 DIM. Mean fluorescence intensity for P (MFIP) and OB (MFIOB), and the shift of percentage of active cells for P (PP) and OB (POB) were measured in isolated, stimulated neutrophils via flow cytometry. Outcomes were assessed with mixed linear regression models accounting for repeated measures. There were no significant differences between treatments in the 4 neutrophil function outcomes. Although MFIOB varied over time, there were no interactions of treatment with time for any outcome. iCa and tCa did not differ between NEG and CON. The LSM ± SE for iCa were: −7 DIM, 1.23 ± 0.02 vs. 1.21 ± 0.02; 1 DIM, 1.07 ± 0.02 vs. 1.02 ± 0.02; 4 DIM, 1.16 ± 0.02 vs. 1.17 ± 0.02 mmol/L, respectively; and for tCa: −7 DIM 2.39 ± 0.04 vs 2.44 ± 0.05; 1 DIM, 2.01 ± 0.04 vs 1.97 ± 0.05; 4 DIM, 2.33 ± 0.04 vs 2.32 ± 0.05 mmol/L, respectively. The proportion of blood samples with tCa <2.15mmol/L at −7, 1 and 4 DIM was 5%, 76%, and 13%, respectively, with no differences between NEG and CON (P > 0.7). However, iCa tended to be weakly positively correlated with MFIOB at −7 DIM. There was no association between tCa and P or OB. We did not observe the hypothesized differences in aspects of innate immunity in multiparous cows fed a negative DCAD.