Laboratory-based confirmation was performed on an enema sample from a suspected case of infant botulism.Methods Clostridium botulinum isolation and toxicity determination by mouse assay were carried out. Results Mice injected with the enema TPGY culture supernatant showed the typical signs of botulism including irritable, dyspnea, bellows breathing and quadriplegia before death. Mice injected with a trypsinilzed culture died, but those injected with the culture heated at 100 ℃ survived. The polyvalent botulinum neurotoxins (BoNTs) antitoxin and the monovalent BoNT type B antitoxin could protect mice from death. Also, Clostridium was isolated from the enema sample and further characterized by morphological characteristics, gram's stain, microscopic, biochemical test and detection of gene producing BoNT toxin by PCR. The result showed that Clostridium isolated from enema sample was C.botulinum that harbors gene producing type B BoNT.Conclusion The patient was associated with type B botulinum toxin poisoning.