Equine trainers generally use either positive or negative reinforcement training procedures to educate their animals and we suggest that these two strategies may have a different impact on the Human-equine relationship (HER). The aim of this study was to compare the effects of positive vs. negative reinforcement during training on the HER. The experimental protocol was conducted in two riding schools with a total of 21 ponies. All animals were trained to walk backwards on a vocal command, using either positive (riding school 1 [RS1]: n=5; riding school 2 [RS2]: n=6) or negative (RS1: n=5; RS2: n=5) reinforcement techniques. Positive reinforcement was a food reward: the experimenter stood in front of the pony, gave the order and waited for the pony to walk backwards; never applying pressure on the lead rope and negative reinforcement was applied by agitating a stick in front of the animal’s head. On all training days, heart rate was measured (R-R Intervals) and behaviour was recorded. HER was assessed by performing a ‘motionless person test’ before and after the training period. Non-parametric Mann-Whitney U-tests were used to compare experimental groups. There were no differences in any of the variables before the training started. However, post training, ponies trained with positive reinforcement were faster to approach a motionless person than the ones trained with negative reinforcement (RS1: 14.6s.±8.9 vs. 146.2s.±65.37, p<0.05; RS2: 50.5s.±33 vs. 300s.±0, p<0.005, respectively). They also spent more time close to this person (distance<1m.: RS1: 95.4% vs. 21.9%, p<0.05; RS2: 70% vs. 0%, p<0.005, respectively) and displayed positive behaviours such as sniffing (RS1: 55% vs. 6.4%, p<0.01; RS2: 20% vs. 0%, p<0.005) and nibbling (RS2: 20.3% vs. 0%, p<0.005) the experimenter. The use of positive reinforcement, contrarily to negative reinforcement, in equine training programmes appears to have beneficial effects on the HER.