l-Glutamine supplementation improves gastrointestinal and immune function in dairy calves during controlled immune and stress challenges. However, it is unknown whether supplementing milk replacer (MR) with l-glutamine improves preweaning dairy calf health and welfare under production conditions. Therefore, the study objective was to evaluate the effects of supplementing MR with l-glutamine on gastrointestinal permeability, immune function, growth performance, postabsorptive metabolic biomarkers, and physiological stress response in preweaning dairy calves. In 3 repetitions, Holstein heifer calves (n = 30; 1.5 ± 0.5 d old; 37.1 ± 0.86 kg body weight) were blocked by serum total protein, body weight, and age, and provided MR (3.8 L/calf per d; 24% CP, 17% fat, 12.5% solids) supplemented with l-glutamine (GLN; 10g/kg MR powder; n = 5 calves/repetition) or nonsupplemented (NSMR; n = 5 calves/repetition). Calves were individually housed with ad libitum starter grain and water access until weaning (56.4 ± 0.5 d old). At 1 and 6 wk of age, urinary catheters were placed, and calves were orally dosed with 1 L of chromium (Cr)-EDTA. Urine samples were collected over a 24-h period for Cr output analysis as an in vivo biomarker of gastrointestinal permeability. Blood was collected on study d 1, 5, 7, 14, 21, 42, and 56 to measure white blood cell counts, cortisol, insulin, glucose, nonesterified fatty acids, serum amyloid A, haptoglobin, and neutrophil: lymphocytes. Two study intervals were used in the statistical analyses, representing greater (P1; wk 1-3) and reduced (P2; wk 4-8) enteric disease susceptibility. Data were analyzed using PROC GLIMMIX in SAS 9.4 (SAS Institute Inc.) with calf as the experimental unit. Overall, total urinary Cr output was reduced in GLN versus NSMR calves. Total Cr output was reduced at 1 wk of age in GLN versus NSMR calves, but no differences were detected at 6 wk of age. Neutrophil:lymphocyte was decreased both overall and during P2 in GLN versus NSMR calves, and neutrophil counts tended to be reduced in GLN versus NSMR calves during P2. No MR treatment differences were detected for average daily feed intake, average daily gain, body measurements, postabsorptive metabolic biomarkers, disease scores, and therapeutic treatments between GLN and NSMR calves. In summary, l-glutamine supplementation reduced gastrointestinal permeability and biomarkers of physiological stress in preweaning Holstein heifer calves.