Zearalenone (ZEN) is produced by Fusarium spp. and is widely found in moldy wheat, corn, and other grains. ZEN has a strong toxicity and causes reproductive and immune disorders and estrogenic syndrome in animals and humans. Biodegradation has been demonstrated as an efficient way to control the hazardous effect of ZEN. A promising way to apply biodegradation in feed is to introduce anaerobic ZEN-degrading microorganisms, which can function during the digestion process in animal intestines. The aim of this study was to isolate anaerobic ZEN-degrading bacteria from anaerobic environments. A strain named F39 was isolated from animal intestinal contents and had a ZEN-degradation rate of 87.35% in 48 h to form trace amount of α- and β-zearalenol. Based on the morphological and physiological properties and phylogenetic analysis of 16S rRNA and rpoB gene sequences, F39 was identified as Clostridium sporogenes. The optimum temperature for the growth of F39 was 37 °C, the optimum pH was 7.0, and the most suitable carbon source was beef extract, while the optimal conditions for the degradation of ZEN were as follows: 35 °C, pH 7.0, and GAM medium. ZEN was degraded by F39 with a high efficiency in the concentration range of 1–15 mg/L. The bioactive factors responsible for ZEN degradation were mainly distributed intracellularly. F39 can degrade most of the ZEN present, but a small amount is broken down into two secondary metabolites, α- and β-zearalenol, and the toxicity of the degradation products is reduced. With an efficiency of 49%, F39 can more effectively degrade ZEN in wheat-based feedstuffs than in other feedstuff, and the degradation efficiency was pH related. To the best of our knowledge, this is the first report of Clostridium sporogenes F39’s ability to maintain the biodegradation potentials.