Mammary-derived serotonin has been implicated in breast-to-bone communication during lactation by increasing parathyroid hormone related-protein (PTHrP) in the mammary gland. It is well established that PTHrP acts on the bone to liberate calcium for milk synthesis during lactation; however, the mechanism of serotonin's regulation of PTHrP has not been fully elucidated. Recently, serotonylation has been shown to be involved in a variety of physiological processes mediated by serotonin. Therefore, we investigated whether serotonylation is involved in serotonin's regulation of PTHrP in the mammary gland using lactogenically differentiated mouse mammary epithelial cells. We investigated the effect of increased intracellular serotonin using the antidepressant fluoxetine or 5-hydroxytryptophan (serotonin precursor), with or without transglutaminase inhibition and the corresponding action on PTHrP induction and activity. Treatment with fluoxetine or 5-hydroxytryptophan significantly increased intracellular serotonin concentrations and subsequently increased PTHrP gene expression, which was reduced with transglutaminase inhibition. Furthermore, we determined that transglutaminase activity is increased with lactogenic differentiation and 5-hydroxytryptophan or fluoxetine treatment. We investigated whether RhoA, Rac1, and Rab4 were potential serotonylation target proteins. We speculate that RhoA is potentially a serotonylation target protein. Our data suggest that serotonin regulates PTHrP induction in part through the process of serotonylation under lactogenic conditions in mouse mammary epithelial cells.