Enterobacter kobei is the species of the Enterobacter cloacae complex, which is phenotypically most closely related to the species E. cloacae. This is the first report of infection caused by a new biotype of E. kobei. A patient with a history of urinary bladder operation developed a urosepsis with an Enterobacter isolate displaying the E. cloacae phenotype. The isolate was classified to the species E. kobei by sequence analysis of the 16S-rDNA, 4 protein-coding genes and enterobacterial repetitive intergenic consensus (ERIC)-cluster analysis. E. kobei was originally described to be Voges-Proskauer (VP) negative. However, the isolates of the present case were VP-positive. After analyzing 120 biochemical tests included in the API20E and the Biotype 100 systems, 4 biochemical tests were identified potentially differentiating this new biotype from E. cloacae.