In the past decade, the withdrawal of antibiotics used as growth promoters (AGP) has increased some poultry industry challenges, such as the rise of intestinal diseases. Experts advocate that AGPs improve performance due to the modulation of the intestinal microbiota, with resulting anti-inflammatory effects. However, the impact and interactions of AGPs with the host intestinal immune system are still unknown, which represents issues in developing effective alternatives for AGPs. Therefore, this study was aimed at better understanding the potential mechanism of action of bacitracin used as AGP and its impacts on the intestinal immune system. Ninety day-of-hatch chickens were randomly assigned to two treatments with three repetitions of fifteen birds, a control (CNT) group with a corn/soybean meal standard diet, and a control diet supplemented with 50 g/ton of feed of bacitracin (BMD). The cytokines’ and chemokines’ production (IFN-α, IFN-γ, IL-16, IL-10, IL-21, IL-6, M-CSF, MIP-3α, MIP-1β, VEGF and CCL-5) were assessed in the jejunum and ileum at 14, 21, 28 and 36 days of age by using a chicken-specific cytokine/chemokine peptide ELISA array. Broilers with BMD supplementation were found to have a lower production of IL-16, IFN-γ, M-CSF, IL-21, MIP-1β and VEGF in the jejunum at 14 d. However, from 21 through 36 days, the effect of BMD on cytokine production in the jejunum was negligible except for CCL-5, which was reduced at D36. In the ileum, BMD effects on the cytokine profile started at 28 d, when BMD-supplemented broilers showed a reduced IL-6 production level. At day 36, BMD reduced IL-16 and MIP-3α production but increased VEGF concentration in the ileum tissue. The present study demonstrated that the use of bacitracin as an AGP modulates the small intestine immune system, especially in the first phase of the broiler’s life (up to 14 days). Moreover, BMD anti-inflammatory effects include not only innate immunity but also seemed to influence the development of the adaptive immune response as seen by the decreased production of IL-21 and IL-16. These results demonstrate that a commonly used AGP in broiler feed had a local anti-inflammatory effect.