Vitamin A mediates many important biological functions in humans and animals. Presence of vitamin A receptors on immune system cells emphasizes their role in immune functions. To assess the effects of in ovo inoculation of vitamin A on the immune system of chicken embryos, 18 days old embryonated eggs were inoculated with 3 different concentrations of retinoic acid (the active form of vitamin A) at 30, 90, and 270 μmol/egg via the amniotic sac. After 6, 18, and 24 h, the spleen and bursa of the embryos were collected for RNA extraction and real-time polymerase chain reaction. The results were dose dependant. After 24 h, inoculation with 270 μmol/egg downregulated relative expression of interferon IFN-α, IFN-β, IFN-γ, interleukin (IL)-1β, IL-2, CXCLi2, IL-12, and IL-13 compared to control in the spleen, indicating an anti-inflammatory effect at this concentration. In comparison, 90 μmol/egg induced greater expressions of the above genes at the same timepoint compared to the 270 μmol. The results of this study indicate that in ovo inoculation of vitamin A can modulate immune functions of the chicken embryo, which might be beneficial for induction of immune responses by in ovo vaccines.