Administration of exogenous glucagon-like peptide 2 (GLP-2) has recently been shown to be a strategy to improve gastrointestinal tract health in dairy calves. However, this strategy may not be a feasible approach in the current dairy calf production system because of practical application and animal welfare issues. An alternative approach is to stimulate the release of endogenous GLP-2 through ingestion of nutrients. Thus, the objective of this study was to evaluate effects of pulse-dose intraruminal butyrate infusion on dynamic change in plasma GLP-2 concentration in dairy calves. Five rumen-cannulated male calves (330 ± 16.0 kg) were used in a 5 × 5 Latin square design with 4-d periods. On d 1 of each period at 0800 h, calves were ruminally infused with 1 of 5 treatments: 0 (saline), 0.3, 0.6, 0.9, and 1.2 g butyrate per kg of BW. Infusion of butyrate was preceded by 12 h of fasting to ensure a steady-state condition. Sequential blood and rumen fluid samples after infusion were taken on d 1 of each period. Data were analyzed using the MIXED procedure of SAS where the model included fixed effect of period, square, treatment, time, and interaction of treatment with time, and the random effect of calf within square. The effects of treatment (pulse-dose of butyrate infusion) were further analyzed using the orthogonal polynomial contrast. Rumen butyrate (P < 0.001) and total VFA (P < 0.001) concentrations increased linearly in a dose-dependent manner while propionate (P = 0.047) and iso-butyrate (P = 0.040) increased quadratically. There was a cubic effect of dose of butyrate infusion on plasma GLP-2 concentration (1.59, 2.15, 0.93, 1.62 and 1.68 ng/mL; P < 0.001); whereas, the concentrations of rumen (P < 0.001) and plasma (P < 0.015) butyrate, and BHB (P < 0.001) increased linearly. Rumen and plasma concentrations of butyrate, BHB, glucose, GLP-2, and insulin were all affected by time (P < 0.001) and interaction of time with treatment (P < 0.001). Plasma GLP-2 was not correlated with BHB, plasma or rumen fluid butyrate levels. This study provides a basis for future calf studies investigating the mechanisms of action of butyrate and other biomolecules to stimulate GLP-2 secretion.