Prophylactic calcium supplementation immediately after calving is a common strategy to prevent clinical and subclinical hypocalcemia in parturient dairy cows. The objective of this study was to evaluate the effect of prophylactic administration of Theracalcium on blood calcium concentration at 24 and 48 h after treatment, in cows without clinical hypocalcemia. Cows (n = 128) from 4 farms were blocked by parity and randomly assigned to receive either calcium gluconate (35% w/v) in combination with calcium glucoheptonate (10% w/v; Theracalcium, Vétoquinol Canada Inc., Lavaltrie, Quebec) or a placebo (medication vehicle solution with no calcium) at first contact with each cow after calving and again 12–24 h later when available for lockup. Each dose was 120mL injected subcutaneously over 2 sites. Total serum calcium concentration (tCa) was measured from coccygeal blood samples before (time 0) and 24 and 48 h after first treatment. There was no significant difference in tCa at time 0 (2.05 ± 0.02) between groups (P = 0.18). Serum tCa at time 0 was highly correlated with parity (r = −0.7) therefore pre-treatment tCa but not parity was used as a covariate in the models. A mixed model was constructed to measure tCa at 24 h after first treatment. For cows that had received 1 injection of calcium before the blood sample at 24 h (n = 95), tCa was significantly higher in the treated cows (P = 0.01): mean ± SE 2.03 ± 0.03 versus 1.90 ± 0.03 mmol/L, accounting for tCa at time 0 and a treatment by tCa at time 0 interaction. At 48 h there was no significant difference in tCa between treatment and control (mean ± SE 2.12 ± 0.02 and 2.10 ± 0.03 mmol/L, respectively) accounting for tCa at time 0 and farm as a fixed effect. With this subcutaneous prophylactic calcium treatment regimen, blood calcium levels were temporarily increased at 24 h after treatment. Further studies of disease and production in the postpartum period are required to determine if such a rise in blood calcium translates into improvements in transition cow health.