The onset of lactation is characterized by marked increases in calcium (Ca) metabolism. Recently emphasis was placed on understanding the profile and dynamics of blood Ca and serotonin in the peripartal cow in response to this change using a randomized 2x2 factorial design. The aims of our study were to determine i) how a prepartum dietary cation anion difference (DCAD) diet and the magnitude of Ca decline at the onset of lactation alter circulating blood serotonin and iCa concentrations dynamics in the periparturient cow, and ii) the relationship Ca versus serotonin during the peripartal period. Thirty-two multiparous Holstein cows were blocked by parity (2 vs > 2) to ensure equal parity number across the experiment, previous 305 d milk yield and expected parturition date and randomly allocated to either positive (+120 mEq/kg; +DCAD) or negative (-120 mEq/kg; -DCAD) DCAD diets from 251 d of gestation until parturition (n = 16/diet). Immediately after calving cows were continuously infused for 24 h with i) an intravenous solution of 10% dextrose or ii) calcium gluconate (CaGlc) to maintain blood ionized Ca concentrations at 1.2 mM (normocalcemia); forming the following 4 treatment groups: negative DCAD and CaGlc (NCa), negative DCAD and dextrose (NDex), positive DCAD and CaGlc (PCa), and positive DCAD and dextrose (PDex) (n = 8/treatment). Blood was sampled every 6 h from 102 h before parturition until 96 h post parturition. During the continuous infusion period (the 24 h immediately following parturition) cows were sampled every 30 min. Cows fed a -DCAD diet prepartum had greater circulating serotonin concentrations pre and postpartum. Time series analyses demonstrated that the pooled mean effect size (ES) of the relationship between ionized Ca and serotonin 36 h before parturition was significantly positive (ES = 0.164) prepartum. We also observed that the overall pooled mean ES of the relationship between prepartum blood pH 24 h prior and serotonin was significantly positive (ES = 0.111); however, for individual treatment groups, relationships were identified between blood pH and serotonin 18 and 6 h before only for the NDex group. Our data suggest that serotonin is an important factor in the regulation of Ca homeostasis during the prepartal period and that the mechanism may be integrated with blood pH.