The effects of sodium butyrate (SB) supplementation in whole milk (WM), and/or a starter mixture (SM) on the growth performance, selected blood parameters, gastrointestinal tract (GIT) development, and tight junction (TJ) proteins of the gastrointestinal tract in preweaned calves were determined. Eighty calves, averaging of 3 (± 0.74) d old, were randomly allocated into 1 of 4 groups (n = 20 per group) and were fed (1) WM and SM without SB (control group); (2) SM supplemented with SB (0.3% of dry matter basis; SM group); (3) WM supplemented with SB (WM, 8.8 g/d) (WM group); or (4) WM and SM supplemented with SB (SMWM group). At d 74, four calves from each group were euthanized for in-depth analysis. The addition of SB to the SM increased the ADG and reduced fecal scores throughout the preweaned period (P < 0.05). SB supplementation in WM notably lowered diarrhea incidence throughout the preweaned period (P < 0.05). The addition of SB to the SM increased blood beta-hydroxybutyric acid and diamine oxidase concentrations at d 24 and insulin-like growth factor 1 concentrations at d 44 (P < 0.05). SB addition to WM prominently enhanced rumen papilla dimensions but reduced jejunum crypt depth alongside villus height and depth (P < 0.05). The addition of SB to the SM diminished the mucous layer thickness of the rumen and increased TJ protein expression in the GIT (P < 0.05). Based on these results, we found that the addition of SB to the SM and WM had a positive effect on calf general health, blood indexes, and GIT development. The addition of SB to both WM and SM could be recommended for preweaned calves.