Acinetobacter lwoffii infections are largely concentrated in mammals; very few reports are available for such infections in fish. In this study, a Gram-negative rod bacillus was isolated from the intestine of grass carp (Ctenopharyngodon idella), and designed as strain CY1. The isolated strain was then identified as A. lwoffii according to morphological, physiological, and biochemical properties, and 16S rDNA sequencing. The phylogenetic tree constructed on the basis of 16S rDNA sequences indicated that strain CY1 is most closely related to A. lwoffii, and has 98.08% similarity with type strain ATCC17925. Experimental infection assays were conducted, and a cumulative mortality rate of 75% was observed in grass carp. Antimicrobial susceptibility test showed that the isolated strain CY1 was highly susceptible to most antimicrobial agents tested, including aminoglycosides, β-lactams, and quinolones. To our knowledge, this is the first report on pathogenic A. lwoffii in grass carp.The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden.