We have successfully conjugated mesalamine (5-aminosalicylic acid, 5-ASA) with xylan, a biopolymer isolated from pineapple stem waste, to form xylan-5-ASA conjugate. The biopolymer was used to provide colon-targeting properties for 5-ASA, a golden standard anti-inflammatory agent commonly used for ulcerative colitis treatment. A series of data from FTIR spectroscopy, UV-Vis spectrophotometry, and HPLC confirmed the xylan-5-ASA conjugate formation. To ensure successful colon targeting properties, in vitro and in vivo drug release studies after oral administration of xylan-5-ASA conjugate to Wistar rats were performed. Xylan-5-ASA conjugate was able to retain 5-ASA release in the upper gastrointestinal tract fluid simulation but rapidly released 5-ASA in the rat colon fluid simulation. In vivo release profile shows a very low peak plasma concentration, reached at 6 h after xylan-5-ASA conjugate administration. The delayed release and the lower bioavailability of 5-ASA from xylan-5-ASA conjugate administration compared to free 5-ASA administration confirmed the successful local colon delivery of 5-ASA using xylan-5-ASA conjugate. The administration of xylan-5-ASA conjugate also exhibited greater efficacy in recovering 2,4,6-trinitrobenzene sulfonic acid-induced colon ulcer compared to free 5-ASA administration. Taken together, xylan isolated from pineapple stem waste is promising to obtain colon targeting property for 5-ASA.