Horse transport is considered a cause of stress in animals and is known to affect the 5-HT concentrations in both the brain and other tissues. The aim of this research was to investigate the effect of horse transportation and slaughter stress on plasma serotonin's concentration and the expression levels of the related 5-HT1B and 5-HT2A receptors in PBMCs. Furthermore, the IL-12 levels and a variety of blood parameters, including triglycerides, total cholesterol, glucose, aspartate aminotransferase, creatine phosphokinase, lactate dehydrogenase, were also considered. This research was carried out on 32 horses submitted to short road transport of 40 km to slaughter. Blood samples were collected in baseline conditions (T0) and 24 h later, after they were slaughtered (T1). The results showed a significantly increased expression of 5-HT1B and 5-HT2A receptors and a significantly decreased expression of IL-12 in PBMCs at T1 vs. T0. Furthermore, a significant increase in cortisol and glucose concentrations, and LDH activity was observed at T1. In contrast, a significantly lower circulating 5-HT concentration was observed at T1 vs. T0. These results indicate that the stress induced by transport and slaughter stimuli led to the serotoninergic system's activation, suggesting that the expression of serotonin receptors could be used as a pivotal marker of stress, with potential applications for the improvement of elective protocols to observe the guidelines relating to transported horses.