A bacterium (designated SM04) which can rapidly grow on zearalenone (ZEN) as sole carbon and energy source was isolated from agricultural soil. On the basis of 16S rDNA sequencing analysis, strain SM04 was classified as a bacterium belonging to the Acinetobacter genus. In this study, the biodegradation of ZEN by the extracellular extracts of strain SM04 liquid cultures in M1 medium and Nutrient Broth medium was examined using HPLC analysis, APCI-MS analysis, and MTT (tetrazolium salt) cell proliferation assay. Results showed no ZEN and other equally estrogenic metabolites were found after 12 h when ZEN was treated with the extracellular extracts of M1 cultures, but no significant (P < 0.01) reduction of ZEN was observed over the 12-h incubation period in the extracellular extracts of Nutrient Broth cultures. Results also indicated that some proteins in the extracellular extracts of M1 cultures were essential to ZEN degradation. The proteins in the extracellular extracts of M1 cultures and Nutrient Broth cultures were analyzed with SDS-PAGE, bands showing different intensities among the two extracellular extracts were processed for protein identification by MALDI-TOF/TOF/MS, and nine proteins from M1 cultures matched the database for Acinetobacter genus with great confidence. Furthermore, the function of some proteins identified is unknown or unconfirmed because of the lack of well-annotated genomic sequence data and protein data for Acinetobacter genus on the public database, but in further studies these data of proteins identified will be useful for screening the genes related to ZEN degradation.