The aim of the present study was to evaluate the effect of different postpartum milking strategies on plasma Ca concentration in multiparous dairy cows. A total of 83 Jersey and Jersey × Holstein crossbreed cows of 2nd to 8th parity, were enrolled in the study before 1st postpartum milking. Milking strategies implemented during the first 2 d postpartum were: once-a-day milking (1M; cows were milked every 24 h; n = 24), twice-a-day milking (2M; cows were milked every 12 h; n = 21), delayed milking (DM; cows were not milked for the first 2 postpartum milkings and were milked every 12 h afterward; n = 19), and restricted milking (RM; cows were milked 3 L every 12 h; n = 19). Blood samples for total plasma Ca analysis were collected from the coccygeal vessels into heparinized vacuum tubes starting before 1st postpartum milking, every 4 h up to 48 h and at 72 h postpartum. Plasma Ca concentration changes during the study period and risk of subclinical hypocalcemia (SCH; Ca ≤ 2.12 mmol/L) at 48 and 72 h postpartum were evaluated using MIXED and GENMOD procedures of SAS, respectively. Prevalence of SCH before 1st postpartum milking was 48%. There were effects of treatment (P = 0.03), parity (P = 0.003), time (P < 0.001), initial calcemic status (P < 0.001; normocalcemic or subclinically hypocalcemic) and time by initial calcemic status (P < 0.001) on plasma Ca concentration. Overall, lower plasma Ca concentration was observed for 2M cows (2.04 mmol/l) compared with DM (2.17 mmol/L; P = 0.04) and RM cows (2.17 mmol/L; P = 0.04), but no differences were observed with 1M (2.12 mmol/l). At 48 h postpartum the risk of SCH was lower for 1M (Risk ratio; RR = 0.27; P < 0.001), DM (RR = 0.55; P = 0.02) and RM cows (RR = 0.41; P < 0.001) than for 2M cows. At 72 h postpartum the risk of SCH was lower for 1M (RR = 0.26; P < 0.001) and DM cows (RR = 0.41; P < 0.001) than for 2M cows. Our results suggest that postpartum plasma Ca concentration and risk of SCH may be influenced by postpartum milking strategies, warranting its study as a prophylactic strategy for hypocalcemia. Project funded by USDA-NIFA (1013457 CFAH).