Abstract Adding antioxidants to semen extender has become one of the major strategies to reduce the damage caused by freezing to stallion sperm. Isoespintanol (Iso) is a natural antioxidant that is known as a good free radical scavenger. The aim of this research was to evaluate the effect of supplementation of freezing media with Iso on functional integrity of equine semen. Semen from five Colombian Creole horses was frozen with 0, 10, 20, 30, 40 and 50 µM Iso. Post-thawing motility and kinetics, structural membrane integrity (SMI), normal morphology (NM) and functional membrane integrity (FMI) of sperm were evaluated. Seminal quality (SQi) and seminal velocity (SVi) indexes were calculated. Through a flow cytometric analysis, mitochondrial membrane potential (ΔΨM), viability and DNA integrity of frozen-thawed semen were assessed. General linear models were adjusted, and means comparisons were performed using Tukey’s test. The significance level was P<0.05. No differences for post-thaw sperm quality parameters were found, except for total motility, which showed a reduction by adding 40 µM Iso (P<0.05). However, the estimation of indexes, showed that supplementation with 20 or 30 µM Iso before semen freezing, can increase post-thaw SQi and SVi values of some horses. A higher proportion of high-∆ΨM sperm for 20 µM Iso was found. It is concluded that Iso can improve the quality and mitochondrial activity of frozen-thawed equine semen, however its effect can vary according to the horse and in high concentrations it could have detrimental effects.