Changes in immune factors expressed by milk somatic cells from Holstein cows with hypocalcemia after calving were investigated in this study. Fourteen multiparous Holstein cows after their 3rd or 4th calving in one farm were used. The cows were divided into 2 groups: 7 cows needing treatment due to onset of hypocalcemia (hypocalcemia group; age = 5.53 ± 0.27 years, parity = 3.14 ± 0.14) and 7 cows without health problems (control group; age = 5.88 ± 0.31 years, parity = 3.57 ± 0.26). Milk samples were collected aseptically using a cannula and mRNA of immune factors expressed by milk somatic cells were analyzed. Milk samples (50 mL) were collected from the right rear mammary gland of cows before milking at day 1 and weeks 1, 2, 4, and 8 after calving. All milk samples showed a negative reaction to the California Mastitis Test. Levels of relative interleukin (IL)-6 and cathelicidin in the hypocalcemia group were lower than those in the control group in weeks 1 to 8. A significant difference in relative IL-6 levels was found in week 4 (P < 0.05). These results suggest that levels of IL-6 expressed by milk somatic cells may be affected by hypocalcemia in dairy cows.