A treadmill is an important tool in the equine analysis of gait, lameness, and hoof balance, as well as for the evaluation of horse rehabilitation or poor performance including dynamic endoscopy. Before all of these uses, horses have to be habituated to a treadmill locomotion. We used principal component analysis to evaluate the relationship between aspects of the horse's temperament and emotional response, and progress in the behavioral habituation to a treadmill. Fourteen horses were tested, by the same familiar handler, using the novel object test, the handling test, and both positive and negative emotional response tests. Then, four stages of gradual habituation of the first work on a treadmill were conducted. Each time, the horse's behavior was filmed. Data obtained from ethograms and heart rate measurements were tested. Four principal components were identified in examined horses: "Flightiness," "Freeziness," "Curiosity," and "Timidity." Flightiness was connected with nervousness, agitation by new objects, and easy excitability, and gradually decreased of features during habituation. Timidity was associated with a lack of courage and stress in new situations, and those features strongly increased when the treadmill was introduced. Freeziness and Curiosity features showed strong stability throughout the whole habituation. The results of this study provide evidence for a connection between temperament, emotional response, and habituation process in a horse.