The aim of this study was to evaluate the effect of centrifugation on the integrity and functionality of equine spermatozoa. Semen of ten stallion of Colombian Creole breed were collected using the artificial vagina method. The stallions were located in El Valle de Aburra (Antioquia, Colombia). Each sperm sample was divided in four aliquots, three of them were centrifuged at one of three forces of centrifugation (600 x g, 1200 x g 1800 x g) during 10 minutes at room temperature, while the fourth was not centrifuged (control treatment). Subsequently, total motility and progressive motility were evaluated by a computer analyzer system (SCA), as well mitochondrial membrane potential, sperm viability and acrosome integrity by JC1, SYBR / PI and FITC/PNA fluorescent assays, respectively. It is concluded that even low levels of centrifugal force, can alter total motility, progressive motility, acrosome integrity and mitochondrial membrane potential of stallion spermatozoa.