Road transport and physical exercise represent stressful stimuli that can lead to homeostasis disruption with direct effect on health status, welfare, and physical performance of the athletic horse. Acupuncture is recognized as a practice modulating the physical well-being of athletes. In this study, the effect of acupuncture treatment on some hematochemical parameters was evaluated in 5 thoroughbred horses after road transport and exercise. Horses competed in 2 official races. For each race, animals were transported from their stables to the racetrack. Horses transported and competed in the first race represent the control group. Two weeks later, the same horses competed in the second race. Before road transport, they were treated with acupuncture (acupuncture group). From animals, blood samples were collected at rest (TPRE); after unloaded (TPOST); 30 minutes after unloaded (TPOST30); at rest in the transit stall (RPRE); at the end of the race (RPOST); and 30 minutes after the race (RPOST30). The effect of transport, exercise, and acupuncture was evaluated on cortisol concentration, white blood cell (WBC) count, and leukocytes population including lymphocytes, neutrophils, monocytes, eosinophils, and basophils. A significant effect of transport (P < 0.01) and exercise (P < 0.001) was found on cortisol, WBC, lymphocyte, and neutrophil values in both groups. Transported and exercised horses subjected to acupuncture treatment showed statistically significant lower cortisol, WBC, and lymphocyte values (P < 0.01). The results found in this study showed that transport and exercise are potential stressors for the athlete horse that may affect its welfare and physical performance. The data suggest that acupuncture practice influences animal's psychological perception of a stressful condition, probably, by modulating the neural, immune, and endocrine control systems.