The objective of this work was to develop a safe and effective delivery vehicle for topical treatment of gemcitabine. The physicochemical properties, drug release rate, drug level in plasma and bladder, and histological changes of tissue after drug administration were investigated. The electrical conductivity, mean size, and viscosity of drug-loaded microemulsions were 0.8-102.0 µS/cm, 116.8-322.5 nm, and 42.9-105.0 cps×10³, respectively. Gemcitabine loaded microemulsions showed a slower and sustained release. After intravesical administration of aqueous control and microemulsions treated, the drug concentrations in plasma were 15.11 µg/ml and 2.81-12.82 µg/ml, respectively, and the accumulation in bladder were 18.27 µg and 9.12-64.16 µg, respectively. Microemulsions slightly decreased the systemic absorption and significantly enhanced the accumulation in bladder tissue. Moreover, the preliminary toxicity studies revealed no overt adverse histological changes or tissue irritation by the microemulsion application. Therefore, the microemulsions were suggested to be a promising drug carrier for intravesical chemotherapy.