Objective of this study was to determine the effects of varying dietary cation-anion differences (DCAD) in prepartum period on milk fever, subclinical hypocalcemia and negative energy balance in dairy cows of Urmia, Iran. Ninety nine Holstein cows assigned to three groups (n=33) were fed diets with DCAD of -24 (anionic), +80 (cationic) and +230 (cationic) mEq/Kg dry matter for 3 weeks before expected calving. Total serum calcium concentration was increased with decreasing DCAD. At calving, mean total calcium concentration in serum was 9.42, 8.44 and 7.87 mg/dl in cows fed diets containing -24, +80 and +230 DCAD respectively. Prevalence of clinical hypocalcemia in cows being fed with -24, +80 and +230 DCAD diets were 0%, 3.03% and 12.12% respectively. Also, prevalence of subclinical hypocalcemia was 9.09%, 15.15% and 27.27% in cows fed -24, +80 and +230 DCAD diets respectively. The prevalence of clinical and subclinical hypocalcemia was lower in cows fed anionic diet than cows fed cationic diets. Prevalence of negative energy balance in cows fed with -24, +80 and +230 DCAD diets was 33.33%, 18.18% and 21.21% respectively. Also, the mean β-hydroxybutyrate in cows fed anionic diet was higher than those fed with cationic diets. There was no significant difference in serum glucose concentrations among the three groups. The prevalence of postpartum negative energy balance was higher in cows fed anionic diet. These findings showed that use of anionic diets during three weeks before calving can protect dairy cows from clinical and subclinical hypocalcemia by increasing the calcium level in serum. To reduce the postpartum negative energy balance, replacement of anionic diet by cationic ions soon after calving is suggested.