Abstract: Effect of penicillin, varying in concentration, on metabolic activity and structural diversity of soil microbial community was analyzed with the Biolog-ECO technology, and changes in the soil microbial community structure were with the DGGE (denaturing gradient gel electrophoresis) technology, so as to explore eco-toxicological effect of penicillin on soil microbial community. PCA based on the BiologEcoplates^TM shows that penicillin high in concentration (400-800 mg·kg^-1) may induce rapid enrichment of microbial groups that use phenolic compounds as carbon source in the soil microbial community. PCR-DGGE analysis reveals that in the soil treated with a high dose(800 mg·kg^-1) of penicillin, Shannon-Wiener diversity index was significantly lowered in the first days and gradual recovered 7 days later with the penicillin in the soil degrading.