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Objective Laboratory investigation was performed on a suspected case of infant botulism. Methods Thirty samples of stool, left-over food and environment swabs related to the case were collected, Clostridium spp. isolation, identification and toxicity determination by mouse assay were carried out, and toxin production for isolate was conducted. Results Mice injected with the stool culture supernatant showed the typical signs of botulism including irritable, dyspnea, bellows breathing and quadriplegia followed by death. The toxicity of the stool culture supernatant was enhanced after the treatment by trypsinization but ceased after being heated at 100℃. The polyvalent antibody against botulinum neurotoxins (BoNTs) and the monovalent antibody against BoNT type E could protect mice from death. One gram-positive Clostridium isolate was cultured from infant stool sample. The morphology of the colony on the Columbia blood agar plate showed characteristics of irregular, translucent and flat with rootlike growth. It was positive for type E BoNT-encoding gene and identified as C. butyricum by 16S rRNA sequencing. Toxin production test illustrated that the C. butyricum isolate could produce type E BoNT. Conclusion This was an infant botulism caused by type E BoNT-producing C. butyricum.