Intrinsic serotonin (5-hydroxytryptamine; 5-HT) synthesized within the mammary epithelium has an important physiological role in milk volume homeostasis in many species including mice, cows, and humans. During lactation, mammary epithelial cells activate 5-HT synthesis by tryptophan hydroxylase 1 (TPH1). TPH1 catalyzes the rate-limiting step in 5-HT biosynthesis within mammary glands. 5-HT synthesized in mammary glands is released into both the apical (milk) and basolateral spaces by a vesicular monoamine transporter. 5-HT released into milk is incorporated by the apical membrane-expressed serotonin reuptake transporter and degraded by the monoamine oxidase A enzyme. Suckling maintains 5-HT at low levels in milk. When the mammary gland becomes filled with milk, 5-HT provides a negative feedback signal that suppresses further milk synthesis in the mammary epithelium. Our research, using human mammary epithelial MCF-12A cells, shows that the expression of β-casein, a differentiation marker, is suppressed via 5-HT-mediated inhibition of signal transducer and activator of transcription 5. Additionally, our results show that reduced β-casein expression in MCF-12A cells is associated with 5-HT7 receptor expression. Furthermore, we show that 5-HT7 receptor-mediated suppression of β-casein expression is involved in the activation of protein kinase A and protein-tyrosine phosphatase 1B. Thus, this mechanism might be associated with the feedback signals by 5-HT within the mammary epithelium. Hence, further research that builds on our findings should include the elucidation of the physiological roles of 5-HT present in milk synthesized by mammary epithelial cells in vivo and its effects on nursing infants.