Objective：To prepare levofloxacin-carboxymethyl-chitosan （LVFX/CMC） microspheres and to study their colon-targeted release in rats. Methods： KromasilRKR100-5C18（250 mm× 4.6 mm）was used as the analytical column and the temperature was maintained at 50℃. The mobile phase consisted of a mixture of acetonitrile： 0. 1 % trifluoroacetic acid （20 ： 80） pumped at a flow rate of 1.0 ml/min, with λex 295 nm and hem 495 nm. Sixty healthy male SD rats were randomized into 2 groups： to receive gastric lavage with normal LVFX （40 mg,control group） and LVFX/CMC microspheres （40 mg,treatment group）. HPLC was used to detect the concentrations of LVFX in the stomach,intestine,cecum,colon and blood in rats in both groups. Results： LVFX was detectable 3 h after LVFX/CMC microsphere lavage. Five hours after lavage,the levels of LVFX in cecum and colon were respectively （3. 394±0. 197） mg and （1. 873±0. 216） mg in treatment group,and （0. 489±0. 123） mg and （0. 078±0. 002） mg in control group （P〈0. 01） . Nine hours after lavage,the levels of LVFX in cecum and colon were （1. 580±0. 234） mg and （0. 713±0. 073） mg in treatment group,and （0. 105±0. 023） mg and （0. 054±0. 016） mg in control group（P〈0.01） ,respectively. Serum level of LVFX peaked at 5 h in treatment group and at 1 h in control group. The concentrations of LVFX were obviously higher in cecum and colon than those in stomach and intestine. Conclusion： The prepared LVFX/CMC microspheres have the property of colon-targeted release in rats.