Objective Laboratory confirmation was performed on 44 stool, food and environment samples from a suspected case of infant botulism. Methods According to the national standard GB 4789.12-2016, Clostridium spp. isolation, identification and botulinum neurotoxins (BoNTs) determination were carried out. Results Mice injected with the culture supernatant of infant’s and mother’s hand swabs, infant’s diaper swab, infant’s stool and enema samples shown the typical signs of botulism before death. The toxicity of culture was enhanced after the treatment by trypsin. The polyvalent BoNTs antitoxin and the monovalent BoNT type E antitoxin could protect mice from death. Also, Clostridium was isolated from the above sample and further characterized by morphological characteristics, virulence gene PCR and strain identification qPCR. The result showed that Clostridium isolated from these samples were C. butyricum that harbors bont/e gene. Conclusion The patient was associated with type E BoNT poisoning that produced by C. butyricum.