Based on the severity of the disease progression, the treatment of ulcerative colitis involves administration of single or multiple therapeutic agents. In refractory cases of ulcerative colitis, FK506 (FK) is the drug of choice in clinics. To maintain a long-term stable blood concentration of FK and to reduce its dosing frequency, we propose single-dose injection of FK-loaded biodegradable microspheres (FK-Ms). FK-Ms were intraperitoneally-injected into mice fed with dextran-sulfate sodium (DSS). Pharmacokinetics study revealed presence of FK in blood for over 20days. The single intraperitoneal injection of the drug-loaded microspheres effectively alleviated DSS-induced murine colitis. Mechanistically, the single injection of FK-Ms inhibited the infiltration of T cells into colon and attenuated differentiation of T cells into interferon-γ secreting Th1 and interleukin-17A secreting Th17 cells in colon-draining mesenteric lymph nodes. Therefore, administration of prolonged-release type microspheres can be considered as an alternative for the effective treatment of inflammatory bowel diseases.