Clostridium butyricum is one of the members of genus Clostridia causing necrotizing enterocolitis in rabbit that characterized by intestinal damage (congestion and patchy hemorrhage in intestinal mucosa) and enlargement of caecum (cecitis). In this study, out of 100 rabbits suffered from severe abdominal distension and diarrhea, 34 isolates suspect to be C. butyricum according to their culture characters and microscopical examinations. Pathogenicity test was performed on susceptible rabbits giving the same symptoms and the same strain was re-isolated again. Suspected isolates were identified by detection of 16S rDNA gene using Polymerase Chain Reaction, sequencing of PCR product and comparing sequencing with Gen Bank and that resulting in 98% homogenicity to C. butyricum. These results confirmed that isolated strain is C. butyricum and could be used in further studies in preparation of vaccine for controlling necrotizing enterocolitis in rabbits.