We analyzed the volatile compounds produced by serotype D Clostridium botulinum (D-CB16) in trypticase peptone/yeast extract/glucose (TYG) medium using gas chromatography/mass spectrometry (GC/MS). The volatile compounds were captured by solid-phase microextraction and applied to GC/MS for separation and identification of the compounds in TYG medium with or without the cultivation of C. botulinum D-CB16. Thirty-five and 34 volatile compounds were identified in media without and with D-CB16 cultivation, respectively. Of the compounds identified in the medium with the strain, twenty-one were not detected in the original medium, indicating that these were produced by C. botulinum D-CB16. Keywords: Clostridium botulinum, Volatile compounds, Gas chromatography/mass spectrometry, Food poisoning, Botulism