The objective of this study was to evaluate the effect of tributyrin (TB) supplementation in milk replacer (MR) on growth performance and plasma glucagon-like peptide-2 (GLP-2) concentration in preweaning dairy calves. Twenty Holstein heifer calves were supplemented with 0.3% palm oil (Control; n = 10) or TB (TB; n = 10) on dry matter basis. The MR supplemented with palm oil or TB, and containing 28.0% CP and 15.0% fat was offered at 578 g/d (dry matter basis) from 7 to 13 d of age, 770 g/d from 14 to 20 d of age, 1,156 g/d from 21 to 41 d of age, 770 g/d from 42 to 48 d of age, and 578 g/d from 49 to 55 d of age, then weaned on 56 d of age. All calves were fed calf starter ad libitum and chopped hay limited 200 g/d (as fed basis). Body weight were measured weekly from 7 to 56 d of age. Blood samples were collected weekly from 7 to 56 d of age and assayed for plasma GLP-2 and metabolite concentrations. A mixed model was used to determine the effects of treatment and day as repeated measures, and their interaction. Average daily gain did not differ between control (0.78 ± 0.03 kg/d; LSM ± SE) and TB (0.72 ± 0.03 kg/d). Dry matter intake (DMI) of MR and hay did not differ between the 2 treatments. However, interaction effects between treatment and day about DMI of calf starter, total DMI and intake of metabolizable energy (ME) were observed (P < 0.05), and these were lower for TB calves at 46, 47, from 50 to 55 d of age compared with control calves. Plasma GLP-2 concentration was higher for TB calves (0.59 ± 0.05 ng/mL) compared with control (0.41 ± 0.05 ng/mL) calves (P < 0.05). Blood glucose and serum β-hydroxybutyric acid concentrations did not differ between the 2 treatments. In conclusion, MR supplemented with TB increased plasma GLP-2 concentration. Despite the decreased ME intake in preweaning dairy calves fed TB, no difference in growth performance was observed which may be related to GLP-2 action on gut development and nutrient absorption.