Azathioprine is a highly efficient immunosuppressant drug used for treatment of inflammatory bowel disease (IBD). Systemic administration of azathioprine results in delayed therapeutic effect and serious adverse reactions. In the current study, we have developed, for the first time, colon-targeted chitosan beads for delivery of azathioprine in colitis rabbit model. Several characterizations were performed for the azathioprine-loaded beads (e.g. drug encapsulation efficiency, drug loading capacity, yield, size, shape and compatibility with other ingredients). The in vitro release profiles of acid-resistant capsules filled with azathioprine-loaded beads showed that most of azathioprine was released in IBD colon simulating medium. The therapeutic effects of azathioprine-loaded beads and azathioprine crude drug were examined on acetic acid-induced colitis rabbit model. Improved therapeutic outcomes were observed in the animals treated with the azathioprine-loaded beads, as compared to the untreated animal controls and the animals treated with the azathioprine free drug, based on the clinical activity score, index of tissue edema, mortality rate, colon macroscopic score and colon histopathological features. In the animals treated with the azathioprine-loaded beads, the levels of the inflammatory mediators, myeloperoxidase enzyme and tumor necrosis factor-α, were significantly reduced to levels similar to those observed in the normal rabbits. Furthermore, the activities of the antioxidant enzymes, superoxide dismutase and catalase, were restored considerably in the animals treated with the drug-loaded beads. The azathioprine-loaded beads developed in the current study might have great potential in the management of IBD.