Food rewards are believed to have a positive valence in horses. The aim of this study was to assess the effect of food rewards on horse behavior before entering a horse chute, and behavior and facial movements while restrained in it. Thirteen female adult horses were brought once daily to an animal handling facility for three weeks. In week 1, baseline period, no reinforcement was applied. In weeks 2 and 3, experimental phase, half of the horses received positive reinforcement treatment after entering and remaining in the chute; the remaining horses were considered as controls (no positive reinforcement applied). There was a cross-over between the groups during the experimental phase. Horses were individually brought to the restraining chute and videos recorded during 60-sec. The duration and number of entries into the area close to the gate leading to the chute were measured before restraining and body posture, neck position, and tail swinging were recorded in the chute. Facial movements were also recorded and scored using EquiFACS methodology. Multilevel linear and logistic models were built to assess behavioral changes from baseline to the treatment phase and between phases (control and positively reinforced). Horses did not change their body posture or tail swings across the different phases (P> 0.1) and were less likely to show lowered neck during the positively reinforced phase (OR: 0.05; CI95%: 0.00–0.56;P= 0.05) compared to baseline. The likelihood of a lowered neck did not differ between the positive reinforcement and control phases (P= 0.11). In the positively reinforced phase, horses seemed to be more attentive (ears forward) and active (less eye closures, more nose movements) than in the control phase. A three-day positive reinforcement phase did not elicit major changes in body behavior in the chute but affected the facial movements of group-housed mares.