Serotonin may be an effective therapy for hypocalcemia. Pre-partum infusions of the serotonin precursor 5-HTP increased circulating calcium in postpartum Holstein dairy cows. However, mammary gland calcium flux in response to 5-HTP infusion has not yet been determined. Elucidation of serotonin’s effects on calcium flux in the transition dairy cow mammary gland may reveal the molecular mechanisms underpinning hypocalcemia. Multiparous Holstein cows were intravenously infused daily with either 1 L of saline (CON; n = 6) or 1 mg/kg bodyweight of 5-hydroxytryptophan (5-HTP; n = 6) for 5.67 ± 0.78 d pre-partum until parturition. Biopsies were performed before treatment, on the d (d1) and d7 after parturition on opposite rear quarters using a punch tool. RNA was extracted from all samples and real-time PCR was performed. Data were analyzed using the delta-delta CT method and statistical analysis was conducted using a 2-way ANOVA for time, treatment, and the interaction on mRNA abundance of calcium sensing receptor (Casr), plasma-membrane ATPase 2 (Pmca2), calcium release-activated calcium channel protein 1 (Orai1), tryptophan hydroxylase 1 (Tph1), serotonin reuptake transporter (Sert), and serotonin receptor 7 (5HTR7). mRNA abundance of Casr was elevated in 5-HTP cows on d1 and d7 (P = 0.0002) compared with CON, and mRNA was more abundant on d1 and d7 postpartum compared with pre-partum (P = 0.001), with a time and treatment interaction (P = 0.007). While Pmca2 abundance increased postpartum compared with pre-partum (P = 0.03), there was no effect of treatment. Orai1 was elevated in CON cows over 5-HTP cows on d1 (P = 0.03), and on d1 and d7 compared with baseline (P = 0.0001), with an interaction of treatment and time (P = 0.03). There was increased abundance of Tph1 (P = 0.03), Sert (P = 0.02), and 5htr7 (P = 0.02) transcripts in 5-HTP compared with CON cows on d1 and d7 postpartum. mRNA abundance of Tph1 (P = 0.03), Sert (P = 0.003), and 5htr7 (P = 0.007) was increased on d1 and d7 relative to pre-partum. These findings demonstrate that 5-HTP infusion pre-partum alters serotonin and calcium axes in the cow mammary gland. We also show these axes are activated postpartum, establishing a molecular profile for calcium and serotonin signaling in early-lactation dairy cows.