Weaning dairy calves from a high milk volume (≥8.0 kg/d) can negatively affect the growth and welfare even if it is performed in a step-down manner. Supplementation of Gln improved gut development of preweaning calves and mitigated weaning stresses of piglets to extents achieved with antibiotics. The study objective was to examine the effect of initiating a step-down weaning scheme with a Gln supplement at an early age on calf starter intake (CSI), average daily gain (ADG), and paracellular permeability of the intestinal epithelium of calves fed a high volume of milk (9.0 kg/d). Thirty-six Holstein heifer calves were assigned to 3 treatments (n = 12) as follows: (1) initiating weaning at 49 d of age (LW), (2) initiating weaning at 35 d of age (EW), and (3) initiating weaning at 35 d with a Gln supplement (2.0% of dry matter intake) from 28 to 42 d of age (EWG). Calves were fed 9.0 kg/d of whole milk until weaning was initiated by abruptly decreasing the milk volume to 3.0 kg/d. Weaning was completed once calves achieved ≥1.0 kg/d of CSI. The paracellular permeability of the intestinal epithelium was assessed with lactulose-to-mannitol ratio (LMR) in the blood on 1 d before, and 3 and 7 d after the initiation of weaning. The blood was analyzed for haptoglobin, lipopolysaccharide-binding protein (LBP), and metabolites including AA. The CSI increased once milk volume was restricted in all treatments. The CSI of LW was greater than that of EW and EWG during the first week of weaning. The LW, EW, and EWG took 11, 19, and 16 d to achieve ≥1.0 kg/d of CSI and were weaned at 60, 54, and 51 d of age, respectively. The body weight (BW) of LW, EW, and EWG at the initiation of weaning were 68.2, 58.7, and 59.5 kg, respectively. Both LW and EWG achieved similar ADG, but ADG of EW was lower than LW during the first week of weaning. All calves had similar ADG during the second week of weaning. The BW of LW, EW, and EWG at weaning were 74.8, 66.5, and 66.4 kg, representing a 2.0, 1.8, and 1.8-fold increase in birth weight, respectively. All calves had similar BW of 88.6 and 164.3 kg at 10 and 20 wk of age, respectively. Regardless of the age, serum haptoglobin and plasma LBP concentrations increased on d 3 and returned to baseline concentrations on d 7 during weaning. The EW had a lower plasma LBP concentration than LW and EWG on d 3 during weaning. The LMR was similar between treatments on d 3 but increased by 44% for EW and LW on d 7, whereas the LMR of EWG remained unchanged during weaning. The postprandial serum concentration of Gln, Met, Trp, and β-hydroxybutyrate were greater for EWG than EW during weaning. Beginning step-down weaning at 35 d with a Gln supplement can help maintain the gut barrier function and wean dairy calves with a satisfactory CSI at 7 wk of age without affecting postweaning growth.