Serotonin (5-HT) is a monoamine that regulates energy balance through the modulation of insulin and lipid metabolism. Here, we hypothesize that manipulating 5-HT pathway by administering Fluoxetine (FLX, a 5-HT reuptake inhibitor) or 5-hydroxytryptophan (5-HTP, a 5-HT precursor), would improve energy metabolism in pre-weaned dairy calves. Bull Holstein calves (21 ± 2 d) were fed milk replacer (8 L/d) with 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. Blood samples were collected before (d-1), daily during treatment administration (d1 to 10) and during a 14-d withdrawal period (at d2, 3, 4, 7, 14) to measure insulin and NEFA concentrations. Calves were euthanized after the 10-d treatment or after the 14-d withdrawal period to harvest pancreas and adipose tissue. Gene expression of 5-HT receptors (5-HTR), 5-HT transporter (SERT) and tryptophan hydroxylase (TPH1) was measured by real-time PCR. Data were analyzed by period using 1- and 2-way ANOVAs in R. Insulin had a treatment by day interaction (P < 0.01), where 5-HTP group had higher circulating concentrations compared with the CON, particularly on d 5, 6, 7 and 9 of treatment; but had only a day effect during withdrawal period (P < 0.001). Circulating NEFA concentrations were not different during treatment or withdrawal period (P > 0.10). After treatment period, adipose gene expression of 5-HT1A tended to be downregulated in the 5-HTP group (P < 0.10), but after withdrawal period 5-HT1F was upregulated by 5-HTP and FLX (P < 0.01) when compared with CON. After treatment period, there was a tendency for downregulation of pancreatic gene expression of SERT­ (P < 0.08) and 5-HT1F (P < 0.10), in FLX and 5-HTP group, respectively, when compared with CON. After withdrawal, serotonin pancreatic receptors were not differentially expressed (P > 0.11). Manipulating serotonin bioavailability of dairy calves increases circulating insulin and modifies the expression of serotonin receptors in the pancreas and adipose tissue.