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The composition of milk replacer (MR) for calves greatly differs from that of bovine whole milk, which may affect gastrointestinal development of young calves. In this light, the objective of the current study was to compare gastrointestinal tract structure and function in response to feeding liquid diets having a same macronutrient profile (e.g., fat, lactose, protein) in calves in the first month of life. Eighteen male Holstein calves (46.6 ± 5.12 kg; 1.4 ± 0.50 d of age at arrival; mean ± standard deviation) were housed individually. Upon arrival, calves were blocked based on age and arrival day, and, within a block, calves were randomly assigned to either a whole milk powder (WP; 26% fat, DM basis, n = 9) or a MR high in fat (25% fat, n = 9) fed 3.0 L 3 times daily (9 L total per day) at 135 g/L through teat buckets. On d 21, gut permeability was assessed with indigestible permeability markers [chromium (Cr)-EDTA, lactulose, and d-mannitol]. On d 32 after arrival, calves were slaughtered. The weight of the total forestomach without contents was greater in WP-fed calves. Furthermore, duodenum and ileum weights were similar between treatment groups, but jejunum and total small intestine weights were greater in WP-fed calves. The surface area of the duodenum and ileum did not differ between treatment groups, but the surface area of the proximal jejunum was greater in calves fed WP. Urinary lactulose and Cr-EDTA recoveries were greater in calves fed WP in the first 6 h post marker administration. Tight junction protein gene expression in the proximal jejunum or ileum did not differ between treatments. The free fatty acid and phospholipid fatty acid profiles in the proximal jejunum and ileum differed between treatments and generally reflected the fatty acid profile of each liquid diet. Feeding WP or MR altered gut permeability and fatty acid composition of the gastrointestinal tract and further investigation are needed to understand the biological relevance of the observed differences., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)