Serotonin, a monoamine derived from l-tryptophan, is naturally produced by peripheral tissues playing a role in several biological functions, including immunity. Here, we tested the safety and effectiveness of administering a serotonin precursor (5-hydroxy-l-tryptophan, 5-HTP) or a serotonin reuptake inhibitor (fluoxetine, FLX) to modulate serotonin bioavailability in dairy calves. 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. Heart rate (HR), respiration rate (RR), rectal temperature (RT), health and fecal scores were recorded daily. Hip height (HH) and body weight (BW) were measured at d-1, 5, and 10. Blood samples were collected at d-1 and daily (d 1–10) to measure serotonin and FLX concentrations. Blood hematology was assessed at d-1 and 10. Data were analyzed using repeated measure or one-way ANOVAs in SAS. Blood serotonin concentrations increased in calves fed 5-HTP starting on d 2, and decreased in calves fed FLX starting on d 6, compared with CON (6325, 2262 vs. 3166 ± 710 ng/mL, respectively, treatment by day interaction; P < 0.001). Health and fecal scores were similar among treatments. BW and HH of all calves increased throughout the experimental period, but calves fed 5-HTP gained more weight compared with CON (0.87 vs. 0.66 ± 0.12 kg/d, P = 0.05). RR was similar between treatments; but feeding 5-HTP lowered RT, while feeding FLX lowered HR, both relative to CON (37.6 vs. 38.0 ± 0.12°C, P = 0.05; and 107 vs. 114 ± 1.9 BPM, P = 0.02). Calves fed 5-HTP and FLX had greater red blood cells counts on d10 compared with CON (7.0, 7.5 vs. 6.3 ± 0.2 × 10⁶/μL, respectively; P < 0.05). FLX fed calves had higher hemoglobin on d10 compared with CON (7.9 vs. 7.3 ± 0.2 g/dL, P < 0.01). There were no differences in total white blood cells counts between treatments, but 5-HTP fed calves tended to have more circulating neutrophils compared with CON (3.4 vs. 2.8 ± 0.3 × 10³/µL, P = 0.06). Manipulation of serotonin pathway by supplementing FLX and 5-HTP is safe and may confer health and growth benefits to Holstein dairy calves.