Abstract Male Holstein calves (n = 80; 42.7 ± 1.44 kg and 14.2 ± 0.81 days of age) were used to evaluate the effects of colostrum consumption and feed restriction on gut functionality in calves subjected to an assembly center simulation (ACS). Well-colostrated (WC) calves received 4L of colostrum within the first 2h after birth and 2L of colostrum in the next 3 feedings, and poor-colostrated (PC) calves received 2L of colostrum within the first 2h after birth. At approximately 14 d of age, calves were subjected to ACS (d-4 to d-1) before a 19h-transport (d0) where they were fed 4L of milk replacer/d (moderate restriction; MO) or 4L of a rehydration solution/d (severe restriction; SV). Treatments were: WCMO, WCSV, PCMO, PCSV and a group of control calves (WCCT) also fed concentrate ad-libitum during ACS and not transported. Twenty-four hours after transportation, a gut permeability test using Cr-EDTA was performed and blood samples for the determination of serum citrulline concentration were collected. Afterwards, one-half of the calves/treatment were euthanized to obtain tissue samples for a gut permeability in vitro analysis and morphometry. Data were analyzed using the MIXED procedure of SAS. Serum Cr-EDTA concentration was greater (P< 0.01) for the WCSV and PCSV compared with the WCMO, PCMO, and WCCT had the least Cr-EDTA concentration after transportation. The PCSV, WCMO and WCSV calves had the least (P< 0.01) serum concentration of citrulline. In vitro measurements of ileum permeability tended (P=0.06) to be greater for PCSV compared with the other treatments. In WCSV, WCMO and PCSV Goblet cells numbers in the ileum were lesser (P=0.02) than in WCCT and PCMO. Feeding rehydrated solution to poor colostrated calves before transport negatively affected gut functionality as indicated by the greatest permeability to Cr-EDTA, the lest serum citrulline concentration, and ileum Globet cells numbers, these findings are supported by the in vitro gut permeability data.