The gastrointestinal tract is continuously exposed to pathogens, thus effective barrier function is important to prevent infiltration. Glucagon-like peptide 2 (GLP2) improves intestinal integrity in various models. Objectives were to characterize effects of GLP2 administration on inflammation and intestinal morphology in feed restricted (FR) cows; a model of leaky gut. Thirteen lactating Holstein cows (153 ± 12 DIM; 732 ± 16 kg BW; parity 2–4) were enrolled in 2 experimental periods. Period 1 (5 d of ad libitum feed intake) served as baseline for period 2 (5 d), during which cows received 1 of 3 treatments: 1) 100% of ad libitum feed intake (AL100; n = 3), 2) 40% of ad libitum feed intake + saline (AL40; 3 mL s.c. 2×/d; n = 5), or 3) 40% of ad libitum feed intake + GLP2 (AL40G; bovine GLP2; Elanco Animal Health, Greenfield, IN; 75 µg/kg BW s.c. 2×/d; n = 5). Milk yield decreased (~34%) similarly in both AL40 and AL40G cows compared with AL100 controls. Relative to AL40, AL40G cows had similar circulating endotoxin and lipopolysaccharide binding protein, decreased haptoglobin (76%; P = 0.01), and tended to have reduced serum amyloid A (57%; P = 0.11). Relative to AL100, AL40 cows had or tended to have decreased jejunum villus width and crypt depth and ileum villus height, crypt depth, mucosal surface area, and cell proliferation (36, 14, 22, 28, 12, and 14%, respectively P ≤ 0.08). Goblet cell area was or tended to be decreased in jejunum, ileum, and colon of AL40 relative to AL100 cows (52, 25, and 17%, respectively; P ≤ 0.06). Compared with AL40, AL40G cows had 46 and 51% increased jejunum and ileum villus height, respectively and increased ileum crypt depth (17%; P ≤ 0.01). In jejunum and ileum, AL40G cows had increased villus height to crypt depth ratios, mucosal surface area, and cellular proliferation (38 and 35%, respectively, 30 and 27%, respectively, and 43 and 36%, respectively; P < 0.01) compared with AL40 cows. Goblet cell area from AL40G cows was increased 59, 17, and 48% in jejunum, ileum, and colon, respectively, relative to AL40 cows (P < 0.01). In conclusion, FR had detrimental effects on intestinal barrier integrity and administrating GLP2 mitigated these negative effects.