The objective of this study was to evaluate the effect of tributyrin (TB) supplementation to milk replacer (MR) on performance, health, and blood concentrations of metabolite and glucagon‐like peptide (GLP‐2) in pre‐weaning calves. Twenty Holstein heifer calves were raised on an intensified nursing program using MR supplemented with either palm oil (CON) or TB (TB) at 0.3% (as fed basis) for 7 weeks starting 1 week after birth. Calves were fed a calf starter and kleingrass from the beginning of the study. Blood samples were obtained weekly to measure blood glucose, serum β‐hydroxybutyric acid (BHBA), insulin‐like growth factor 1 (IGF‐1), and plasma GLP‐2 concentrations. Starter DMI and metabolizable energy (ME) intake were lower in TB calves at 46, 47, from 49 to 55 days after birth compared with the CON calves. However, any growth parameters were not affected by TB treatment. Blood glucose, serum BHBA, and IGF‐1 concentrations were not affected by TB supplementation. On the other hand, mean plasma GLP‐2 concentration among whole experimental period was higher for TB (0.60 ng/ml) compared with CON (0.41 ng/ml). In conclusion, feeding MR supplemented with TB increases plasma GLP‐2 concentration, which might counterbalance the growth performance of TB calves despite the decreased ME intake.