To investigate the release characteristics and cytotoxicity of 5-fluorouracil loaded polylactide microspheres on lung cancer cell lines. The release characteristic of 5-FU-PLA microspheres was detected by constant temperature vibration dialysis assay. The antitumor activities of 5-FU-PLA microspheres against human and mouse lung, gastric and hepatocellular carcinoma cell lines were detected byMTT colorimetric assay. 5-FU-PLA microspheres had a good function of extended-release, its t 1/ 2 was 10. 4 days, and the release process had no blow up effect. The lung and gastric cancer cell lines were more sensitive than hepatocellular cancer cell line to 5-FU-PLA microspheres. The antitumor activity significantly correlated with the time of drug action and the dose of 5-FU released from the microspheres. 5-FU is well distributed in 5-FU-PLA microspheres. Its preparing and releasing processes have no influence on the effect of 5-FU. It can keep the antitumor activities for a relatively long time. The current study provides an experimental basis for interventional therapy of tumors.