Heat stress has negative effects on biological defense mechanisms such as the immune response in chickens, and organs of the immune system, such as the spleen, are atrophied by heat stress in broiler chickens. In order to assess heat stress-induced functional changes of immune mechanisms in the spleen of broiler chickens, expression of genes encoding splenic cytokines (Th1 type, Th2 type, and pro-inflammatory cytokines) was analyzed. Heat exposure at 34°C for 15 days significantly induced the spleen involution, increased interleukin (IL)-4 and IL-12 expression and decreased interferon (IFN)-γ. However, expression of IL-6, 10, 13, and 18 was not affected. Heat stress reduced feed intake, which may affect the spleen weight and cytokine expression. Therefore, a pair-fed group at 24°C (24PF) was included in which chickens received the same amount of feed as those in the group at 34°C. Spleen weight was not affected by the reduction in feed intake. IL-4 expression in the 24PF group was higher than that in the control group. Further, IFN-γ expression increased and IL-12 expression was not affected by the reduction of feed intake, suggesting that the feed intake reduction induced by heat stress does not modulate splenic cytokine expression in broiler chickens. These data suggest that heat stress induces spleen involution and affects the expression of splenic cytokines such as IL-12 and IFN-γ in broiler chickens, but not through the feed intake reduction.