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Vaccinations are used to reduce the severity and duration of future disease in humans and animals by presenting a weakened or killed version of a virus to the immune system. Previous studies show that the immune response can be affected by the concentration of chemical messengers, called cytokines in the blood. Cytokine production has been shown to change throughout the day, so it has been suggested that the immune system would be better able to respond more strongly or quickly at a certain time of day. If this were true, it may be possible to improve vaccine efficacy and improve animal health by simply changing the time of vaccination. The purpose of this study was to determine if time of day of vaccination affects the gene expression of Interleukin 10 (IL-10), and inti-inflammatory cytokine, in horses. Eight Quarter Horse mares (10.5 ± 5.8 yrs) and 6 Miniature Horse geldings (7.0 ± 2.6 yrs) were randomly assigned into an AM or PM vaccination group. Horses in the AM and PM vaccination groups were vaccinated intramuscularly against Eastern and Western encephalomyelitis, rhinopneumonitis (EHV-1 and EHV-4), influenza (type A2), and West Nile Virus at 0700 and 1900 h, respectively. Blood samples were collected via jugular venipuncture on days 0, 7, 14, and 21 for analysis. RNA was extracted from the serum samples and subjected to reverse transcription and quantified using real-time PCR. IL-10 gene expression increased post-vaccination in all horses vaccinated in the AM, except on d 14 in Miniature Horses. IL-10 gene expression was higher in the AM vaccination group, regardless of breed, compared to the PM vaccination group. This study suggests that AM vaccinations administered in the morning hours may elicit an increased immune response.