Serotonin (5-HT) in the mammary gland is known to regulate processes such as calcium homeostasis, tight junction permeability, and milk protein gene expression. The rate-limiting enzyme in the synthesis of non-neuronal 5-HT is tryptophan-hydroxylase (TPH1). Our objective was to further explore the roles of 5-HT in the mammary gland during lactation. We used whole body TPH1 knockout dams (KO; 5-HT deficient, n = 4) and compare them to wild-type (WT; n = 4) and rescue (RC; KO + 100 mg/kg 5-hydroxytryptophan injected daily, n = 4) dams. Mammary tissues were collected on d 10 of lactation. Total RNA extraction, amplification, library preparation, and sequencing were performed following the Illumina mRNA-seq protocol. Sequencing reads were mapped to the mouse reference genome using Tophat. The resulting alignments were used to reconstruct transcript models by Cufflinks. Differential gene expression was analyzed using Cuffdiff. Overall, 97 and 204 genes (false discovery rate, FDR ≤ 0.01) showed at least a 2-fold expression difference between WT and KO and between WT and RC, respectively. Most of the differentially expressed genes (DEG) are related to calcium homeostasis, regulation of apoptosis, cell cycle and cell differentiation, proliferation, and the immune response, among others. Additionally, enrichment analysis using Gene Ontology (GO) and Medical Subject Headings (MeSH) databases revealed the alteration of several biological processes (FRD ≤0.01) including fat cell differentiation and lipid metabolism, regulation of extracellular signal-related kinases and mitogen-activated kinase cascades, insulin resistance, nuclear transport, regulation of membrane potential, release of calcium from the endoplasmic reticulum into the cytosol. The majority of the biological processes and metabolic pathways altered in the KO dams are required for mammary gland homeostasis. Our study reveals the importance of non-neuronal 5-HT for normal mammary gland function and lactation. The potential modes of regulation of the bovine mammary gland during lactation by 5-HT should be further investigated.