Subclinical hypocalcemia (SCH) predisposes cows to other periparturient disorders. Mitigation strategies are crucial to prevent dairy cows from succumbing to SCH. Our lab has previously demonstrated that pre-partum infusions of 5-hydroxytryptophan (5-HTP), the precursor to serotonin, modulates calcium metabolism in transition period dairy cows, yet the precise mechanism in the mammary gland is unknown. The objective of this study was to determine if intravenous (IV) infusion of 5-HTP in mid-late lactation dairy cows regulated calcium metabolism in the mammary gland. This study utilized a randomized complete block design with 12 multiparous Holstein cows, blocked by parity, receiving either IV 1.5 mg/kg 5-HTP (n = 6) or saline (n = 6) for 3 consecutive days. Baseline blood samples were taken before treatment and blood and mammary tissue was collected at 0, 8, and 24 h post final 5-HTP administration. Mammary tissues were analyzed using qPCR to evaluate mRNA expression of calcium sensing receptor (CaSR), parathyroid hormone related-protein (PTHrP), plasma membrane calcium ATPase2 (PMCA2), calcium release-activated calcium channel protein 1 (ORAI1), and secretory pathways calcium-ATPase 1 (SPCA1), and whole blood samples were analyzed for serotonin and total calcium concentrations. Milk yield was recorded daily. Serotonin concentrations were increased (P < 0.0001) in 5-HTP treated cows. Interestingly, there was no difference in CaSR, PTHrP, PMCA2, ORAI1, and SPCA1 expression between 5-HTP and saline treated cows (P = 0.51, P = 0.92, P = 0.43, P = 0.16, and P = 0.08, respectively). However, total blood calcium decreased in 5-HTP compared with saline treated cows after each IV infusion (P = 0.0074, P = 0.0901, P = 0.0008, respectively). Milk yield was not different (P = 0.57) between treatments. This data suggests serotonin’s modulation of calcium homeostasis may be dependent upon physiological stage of lactation in the dairy cow. Further experiments should be conducted to determine the impact of stage of lactation on serotonin’s manipulation of calcium metabolism.