Dairy calves are born with a naïve immune system and are susceptible to metabolic disorders that can lead to impaired growth and increased mortality rates. Serotonin is a monoamine produced by peripheral tissues and has been proposed to act as an immunoregulatory molecule, but its role in immunity is unclear in dairy calves. Hence, we studied the impact of increased serotonin bioavailability on the immune system of dairy calves by administering a serotonin precursor (5-hydroxy-l-tryptophan, 5-HTP) or a serotonin reuptake inhibitor (fluoxetine, FLX). Bull Holstein calves (21 ± 2 d old) were fed milk replacer (8 L/d) with either saline (CON, 8 mL/d, n = 8), FLX (40 mg/d, n = 8) or 5-HTP (90 mg/d, n = 8) for 10 consecutive d in a complete randomized block design. After 10 d of supplementation, peripheral leukocytes were isolated from whole blood and RNA was extracted to measure gene expression of serotonergic components including serotonin receptors (5-HTR), serotonin transporter (SERT), tryptophan hydroxylase (TPH1), amino acid decarboxylase (AADC), monoamine oxidase (MAO), interferon gamma (IFNg) and interleukins (IL) by RT-qPCR using the Fluidgm Delta-Gene Assay. Data were analyzed using one-way ANOVAs in R. All serotonin receptors were expressed in peripheral leukocytes of dairy calves. Oral supplementation of 5-HTP upregulated gene expression of 5-HTR1A, -1B, -1D, -1F, -3B, -3C, and -4 (P < 0.04) and tended to upregulate 5-HT2B and downregulate 5-HT3A (P < 0.06). Fluoxetine supplementation upregulated gene expression of 5-HT2C (P = 0.03) and downregulated 5-HT2A and -3A (P < 0.006). Both treatments upregulated AADC and MAO gene expression (P < 0.03) but no differences were found for TPH1 and SERT expression compared with CON (P > 0.13). Expression of IL-1B, -4, and -12B were upregulated by FLX while expression of IFNg, IL-2, -4, -13, and -17A were upregulated by 5-HTP supplementation (P < 0.04). Serotonin activates transcription of serotonergic components and specific immune markers on peripheral leukocytes, suggesting a possible immunomodulatory role in dairy calves.