Serotonin (5-HT) is a monoamine that play a role in the regulation of energy balance through the modulation of glucose and lipid metabolism. Here, we examined whether manipulating 5-HT pathway by administering Fluoxetine (FLX, a 5-HT reuptake inhibitor) or 5 hydroxy-L-tryptophan (5-HTP, a 5-HT precursor), would impact hepatic glucose metabolism in pre-weaned dairy calves. Bull Holstein calves (21 ± 2d old) 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), during treatment (d1–10) and after treatment withdrawal (d12, 13, 14, 16, 24) to measure glucose concentrations. Two subset of calves (n = 4/group) were euthanized on d10 or d24. Liver tissue was harvested to measure gene expression of 5-HT receptor 2 (5-HTR2A, 2B, 2C), 5-HT transporter (SERT), tryptophan hydroxylase (TPH1), glucose transporters (GLUT-1 to 12) and gluconeogenic enzymes (PDK4, PC, G6P) by real-time PCR. Data were analyzed using mixed models in SAS. Calves fed FLX and 5-HTP had higher circulating glucose compared with CON (110 and 105.5 vs 96 ± 3.8 mg/dl, respectively; P < 0.001). On d10, TPH1 tended to be downregulated in 5-HTP treatment (P < 0.10) and SERT was downregulated in both treatments compared with CON (P < 0.01). On d24, SERT and TPH1 were not different among treatments. On d10, 5-HTR-2A was and 2B tended to be upregulated in the 5-HTP treatment (P < 0.09) while 2C was downregulated in the FLX treatment compared with CON (P < 0.02). On d24, 5-HTR-2A and 2C were not expressed and 2B was not different among treatments. Gluconeogenic enzymes were not different among treatments on d10, but PDK4 was and G6P tended to be downregulated and PC was upregulated in 5-HTP treatment compared with CON (P < 0.07) on d24. On d10, glucose transporters were not different among treatments, while on d24 GLUT-9 tended to be upregulated in 5-HTP treatment and GLUT-12 was downregulated in both treatments compared with CON (P < 0.09). Feeding FLX and 5-HTP increased circulating glucose and altered hepatic expression of gluconeogenic enzymes in dairy calves.