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Vaccines are frequently used in animal health to stimulate humoral and cell-mediated immune responses. The cell-mediated immune response uses specific cells which communicate through messenger molecules, like cytokines, to elicit the response. Previous studies suggest that Th1 cytokine expression should increase when antigen exposure occurs in the evening due to the influence of a circadian rhythm on the immune response. The purpose of this study was to determine if time of day of vaccination influenced the gene expression of interferon gamma (IFN-γ), a Th1 cytokine, in the blood of horses. Eight Quarter Horse mares (10.5 ± 5.8 yrs) and six Miniature Horses (7 ± 2.6 yrs) were randomly assigned to one of two vaccination groups: AM or PM. All horses received mixed grass hay and water ad libitum and were housed in outdoor paddocks with access to shelter. 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, tetanus and West Nile Virus at 0700 or 1900 h, respectively. Blood samples were collected via jugular venipuncture immediately prior to vaccination (d 0) and at 1300 h on d 7, 14 and 21 post-vaccination. RNA was extracted, subjected to reverse transcription and quantitative real-time PCR to determine IFN-γ gene expression levels. IFN-γ gene expression was increased in the AM vaccination group compared the PM group (P > 0.05) on d 7, 14, and 21 post-vaccination. IFN-γ expression in the AM vaccination group was greater on d 7 for both breeds and d 14 for the mares compared to d 0 (P > 0.05). Expression of IFN-γ did not increase post-vaccination in the PM vaccination group. The trend of increased IFN-γ expression in the AM vaccination group suggests that an AM vaccination may be associated with a cell-mediated response.