Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects.