Mitomycin C (MMC) loaded polylactic acid (PLA) microspheres were fabricated by oil-in-water (O/W) single-emulsion solvent evaporation technique. The preparation conditions were optimized. The results indicated that the drug loading rate and encapsulation rate reached maximal values (5.62% and 49.1%) when the ratio of MMC to PLA was 10/90. The morphology of microspheres was observed by scanning electron microscopy. The release tests showed that the microspheres could control release MMC over 30 days and the cumulative release was 84.8% in vitro. Microspheres were co-cultured with mouse NIH-3T3 fibroblast cells and the MTT results showed that the MMC loaded microspheres could effectually inhibit the cell growth.