Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several reproductive processes, i.e., it enhances hyperactivation, motility, and promotes the acrosome reaction in mammalian sperm, but it has not been described in the stallion sperm yet. Therefore, using both immunofluorescence and western blot techniques, we searched for and found some serotonin markers such as 5-HT, 5-HT1B, 5-HT2A, 5-HT₃ receptors, both TPH1 and MAOA enzymes, and serotonin transporter (5-HTT) in stallion sperm. In addition, we found a non-neuroendocrine cell, V-MAT₁ transporter, which has not been previously reported in mammalian sperm. Our results suggest that serotoninergic system is present in stallion sperm, which could be a pharmacological target to increase the conception rates in domestic horses.