Serotonin (5HT) regulates mammary gland physiology and Ca dynamics during lactation. Our lab’s transition period rodent research revealed that 5HT is involved with Ca transport to and within the mammary epithelial cell (MEC). Additionally, 5HT increased parathyroid hormone-related peptide (PTHrP) production and bone mobilization in these rodents. The objective of the following cell culture experiment was to evaluate the effect of 5HT and Ca on PTHrP production and Ca transport in the bovine MEC, providing the first steps in evaluating the 5HT, Ca, and PTHrP axis in bovine mammary epithelium. Immortalized bovine mammary epithelial cells (MAC-T) were grown to 95% confluency and then plated on a 24 well plastic plate coated with Matrigel (n = 3/treatment). Cells were allowed to grow for 6 d in standard growth media. On d 7, cells were treated with lactogenic media only (control), lactogenic media + 5HT (200 µM), lactogenic media + EGTA (0.6 µM), or lactogenic media + 5HT + EGTA. After 48 h, cells were harvested and extracted for qPCR analysis. Expression of β casein was measured to confirm the cells were lactogenic. Relative expression of calcium release-activated calcium channel protein 1 (ORAI1) was increased 0.6-fold in 5HT + EGTA treated cells compared with control (P = 0.06) and 0.8-fold compared with EGTA treated cells (P = 0.03). Relative expression of plasma membrane Ca ATPase 2 (PMCA2) tended to increase in cells treated with 5HT + EGTA compared with control (P = 0.06) while secretory pathway Ca ATPase 1 (SPCA1) tended to increase in cells treated with EGTA (P = 0.07). Relative expression of PTHrP tended to increase in cells treated with 5HT and 5HT + EGTA compared with control (P = 0.06). These results are consistent with our rodent data in that they support a role for 5HT in the transport of Ca to and within the MEC as well as into the milk. In addition, an increase in PTHrP potentiates increased bone resorption, thus increasing Ca mobilization. The apparent synergistic effect of 5HT and EGTA on the expression of some genes warrants further investigation. We believe with additional replication we will provide evidence that 5HT is altering Ca dynamics in the dairy cow.