Serotonin (5-HT) has been shown to be involved in calcium homeostasis, modulating calcium concentration in blood. In addition, 5-HT participates in a variety of metabolic pathways, mainly through the modulation of glucose and lipid metabolism. The hypothesis of the present study was that the prepartum administration of 5-hydroxy-l-tryptophan (5-HTP), a 5-HT precursor, would affect endocrine systems related to calcium homeostasis, and interact with other endocrine and metabolic pathways during the transition period. In this study, 20 Holstein dairy cows were randomly assigned to 2 experimental groups. Both groups received a daily i.v. infusion of 1 L of either 0.9% NaCl (control group; n = 10) or 0.9% NaCl containing 1 mg of 5-HTP/kg of BW (5-HTP group, n = 10). Infusions started d 10 before estimated parturition date and ended the day of parturition, resulting in a minimum of 4 d of infusion (8.4 ± 0.7 d of infusion). Until parturition, blood samples were collected before the daily infusions, and postpartum daily until d 7, and on d 30. Plasma concentrations of parathyroid hormone (PTH) were transiently increased at parturition and on d 1 in control cows. In the 5-HTP group PTH remained unchanged. The concentration of pyridinoline (PYD), an established marker for calcium release from the bone to the bloodstream, increased on d 1 postpartum only in the 5-HTP group. In control cows, PYD concentrations did not change on d 1 postpartum. Melatonin concentrations were slightly but significantly increased in the 5-HTP group compared with the control group. Insulin concentrations decreased in both groups postpartum. Before parturition, leptin concentrations decreased in both groups and remained at this level until d 30 postpartum. Plasma IgG concentrations decreased in both groups on d -1 postpartum. Haptoglobin increased in both groups on d -1 and remained at this level until d 7 postpartum. No differences between groups were observed for insulin, glucagon, IgG, leptin, adiponectin, and haptoglobin concentrations. The results obtained in the present study evidenced that 5-HT is regulating calcium homeostasis independent of PTH. The lack of treatment effects on IgG and on other hormones and metabolites indicates that 5-HTP did not affect these other metabolic pathways and the IgG concentration during the transition period.