Hypocalcemia during the transition period in dairy cows has detrimental effects on animal health, welfare, and production. While clinical hypocalcemia affects 2 to 5% of cows in the US, approximately 50% of cows succumb to subclinical hypocalcemia. Serotonin (5-HT) has been suggested as a therapeutic target for prevention of hypocalcemia. Our objective was to determine the effects of pre-partum intravenous (IV) administration of a 5-HT precursor on calcium homeostasis postpartum in multiparous dairy cows. We hypothesized that the treatment would increase serum calcium and calcium transport into the mammary gland. Twelve (avg. lactation number 3.67 ± 0.43) Holstein cows were IV infused for 5.75 ± 0.82 d pre-partum, beginning approximately 7d before their predicted calving date until calving, with saline (CTL; n = 6) or 1.0 mg/kg 5-hydryoxytryptophan (5-HTP; n = 6), the immediate precursor for 5-HT synthesis. Mammary gland biopsies were performed approximately 2 weeks pre-partum, and d1 and d7 postpartum. Blood and urine were collected daily from the first biopsy through d14 and on d30 of lactation. Colorimetric assays were performed for total calcium in serum and relative mammary mRNA expression was evaluated by RT-PCR. All statistical analysis was performed in SAS using a mixed model ANOVA. Cows infused with 5-HTP had decreased feed intake postpartum compared with CTL (P = 0.0004; 34.75 ± 1.6 kg CTL vs. 30.25 ± 2.8 kg 5-HTP) and overall decreased milk yield (P = 0.0054; 18.35 ± 1.07 kg CTL vs. 17.10 ± 1.04 kg 5-HTP), although colostrum milk yield was not different (P = 0.88). Serum total calcium tended to increase in 5-HTP cows for 14d postpartum (P = 0.07; 2.89 ± 0.09 mM 5-HTP vs. 2.66 ± 0.09 mM CTL). Basolateral mammary epithelial cell calcium sensing receptor (CaSR) mRNA was increased in 5-HTP compared with CTL cows (P = 0.035), as was apical calcium pump plasma membrane calcium ATPase2 (PMCA2) (P = 0.018) on d 1 and d 7 of lactation. These results suggest that 5-HTP treatment prepartum increases postpartum circulating calcium concentrations and calcium transport in the mammary gland.