L-glutamine (GLN) supplementation improves intestinal integrity and immune function in dairy calves during controlled stress challenges. However, it is unknown whether supplementing milk replacer (MR) with GLN offers similar benefits to intestinal integrity and immune function or reduces markers of physiological stress in pre-weaned dairy calves managed under production-relevant conditions. Thirty Holstein heifer calves [1.5 ± 0.5 days old; 37.1 ± 0.9 kg body weight (BW)] were blocked by serum total protein, BW, and age, and provided MR (3.8 L/calf/day, 24% CP, 17% fat, 12.5% solids) supplemented with GLN (10g/kg dry MR; n = 15 calves) or not-supplemented (NS/control; n = 15 calves). Calves were individually housed with ad libitum starter grain and water access until weaning (56.4 ± 0.5 days old). At 2 and 5 weeks of age, urinary catheters were placed, and calves were orally dosed with 1 L Cr-EDTA. Urine samples were collected over a 24-hr period for Cr output analysis as an in vivo biomarker of intestinal permeability. Blood was collected on study days 1, 2, 5, 7, 14, 21, 42, and 56 to measure haptoglobin, serum amyloid A, and neutrophil:lymphocyte. Two study periods were identified representing greater (P1; weeks 1-3) and reduced (P2; weeks 4-8) enteric disease susceptibility. Data were analyzed using PROC GLIMMIX in SAS 9.4 with calf as the experimental unit. Overall, total Cr output was reduced (P < 0.01; 29.4%) in GLN versus NS calves. Total CR output was decreased at 2 weeks (P = 0.04; 42.6%) in GLN versus NS calves, but no differences were detected at 5 weeks. Neutrophil:lymphocyte was decreased (P < 0.01; 28.3%) during P2 in GLN versus NS calves.