Background: Snail (Oncomelania hupensis) control is an important and effective preventive strategy in schistosomiasis control programs, and screening microbial molluscicidal agents is one of the most promising categories in biomolluscicides. Objective: To purify and identify the molluscicidal ingredient (MI) obtained from strain SL-30's exocellular broth. Materials and Methods: The active extracts extracted from SL-30's exocellular broth was purified on a silica gel column guided by molluscicidal activity assay against Oncomelania hupensis, then the MI was obtained. NMR spectroscopy and LC-MS/MS analysis was used to identify the molecular structure of the MI. Results: Molluscicidal activity bioassay showed that the MI exhibited significant molluscicidal activity with the LC 50 values of 0.101, 0.062, and 0.022 mg/L, respectively, in the case of exposure period of 24 h. From 1 H NMR, 13 C NMR, 1 H- 1 H COSY, and 1 H- 13 C HSQC spectra, partial important structure fragment was obtained, and the relative molecular weight of the MI showed 326 according to LC-MS analysis. Then, on these grounds, it was indicated that the molecular structure of the MI had a higher similarity to Gliotoxin with the molecular formula of C 13 H 14 N 2 O 4 S 2 . The quasi-molecular ion of m/z 325.45 was further analyzed by MS 2 as the parent ion, and two daughter ions obtained at m/z 295.11 [M-CH 2 OH]- and m/z 261.08 [M-CH 2 OH -2S]−.Conclusion: The MI was finally confirmed as Gliotoxin.