Technical Abstract: The objective of the current research was to determine if exogenous melatonin would exert a “protective” effect on the gastrointestinal tract of sheep and prevent or reduce the horizontal transfer of E. coli O157:H7 from experimentally-infected to non-infected or “naïve” sheep. Sixteen crossbred ewes were housed indoors and adapted to a high concentrate ration. Ewes were randomly assigned to one of four rooms and treatment (3 ewes/room, 6 ewes/treatment) and received either Control (gelatin capsule only) or Melatonin (5.0 mg/kg BW/d). Four additional ewes served as “carrier” sheep (one/room) and were experimentally-infected via oral gavage with E. coli O157:H7. Three days post-challenge, carrier ewes were housed with naïve sheep and remained with them for the remainder of the experimental period. Treatments were administered to the naïve sheep one day prior to introduction of the carrier sheep and on each of the remaining seven days of the experimental period. Fecal samples were collected via rectal palpation from the carrier sheep daily throughout experiment and from the naïve sheep daily for 5 d, starting two days following introduction of the carriers. On day 8 of the experiment, all ewes were euthanized and tissues from the rumen, ileum, cecum, colon, and rectum, as well as their respective lumen contents, collected. The carrier sheep quickly infected the naïve ewes which had similar fecal concentrations as the carrier animals throughout the 5-d sampling period. Melatonin treatment had no effect (P greater than 0.10) on daily fecal shedding, luminal content concentrations, or in the percentage of GIT tissue positive for the inoculated strain of E. coli O157:H7.