Five nitrogen-fixing bacterial strains (SP1 T , NN143, NN144, NN208 and HX148) were isolated from stem, root or rhizosphere soil of sugar cane (Saccharum officinarum L.) plants. Cells were Gram-negative, motile, rods with peritrichous flagella. DNA G+C content was 55.0±0.5 mol%. Sequence determinations and phylogenetic analysis of 16S rRNA gene and rpoB indicated that the strains were affiliated with the genus Enterobacter and most closely related to E. radicincitans DSM 16656 T and E. oryzae LMG 24251 T . Fluorimetric determination of thermal denaturation temperatures after DNA-DNA hybridization, enterobacterial repetitive intergenic consensus PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiated the whole-genome, genotype and protein profiles from those of E. radicincitans and E. oryzae. The strains' cell fatty acid composition differentiated them from E. radicincitans and E. oryzae by containing a higher level of summed feature 2 (C 16 : 1 v7c and/or C 16 : 1 v6c) and a lower level of C 17 : 0 cyclo. Their physiological and biochemical profiles differentiated them from E. radicincitans by being positive for methyl red test, ornithine decarboxylase and utilization of putrescine, D-arabitol, L-fucose and methyl a-D-glucoside and being negative for arginine dihydrolase, and differentiated them from E. oryzae by being positive for aesculin hydrolysis and utilization of putrescine, D-arabitol and L-rhamnose and being negative for arginine dihydrolase, lysine decarboxylase and utilization of mucate. The five strains therefore represent a novel species, for which the name Enterobacter sacchari sp. nov. is proposed, with the type strain SP1 T (5CGMCC 1.12102 T 5LMG 26783 T ).Both pathogenic and beneficial bacteria to plants have been affiliated with several species of the genus Enterobacter in the family Enterobacteriaceae (Chung et al., 1993; Kämpfer et al., 2005;Madhaiyan et al., 2010;Peng et al., 2009;Zhu et al., 2011). Beneficial nitrogen-fixing bacteria belonging to several genera of the family Enterobacteriaceae have been found and have been shown to fix N 2 in association with sugar cane (Saccharum officinarum L.) worldwide and to promote sugar cane growth (Govindarajan et al., 2007;Lin et al., 2012;Loiret et al., 2004;Magnani et al., 2010;Mehnaz et al., 2010;Mirza et al., 2001;Taulé et al., 2012). Strain SP1 T was isolated from a stem of sugar cane cultivar GT11 grown in Nanning, Guangxi, China in 1994. Sugar cane internodes were surface-sterilized by immersing intoThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SP1 T , HX148, NN143, NN144 and NN208 are JQ001784, HQ204281, HQ204313, HQ204314 and HQ204315, for rpoB sequences are JQ001786, JQ001787, JQ001788, JQ001789 and JQ001790 and for nifH sequences are JQ001785, HQ204226, HQ204258, HQ204259 and HQ204260, respectively. 70 % ethanol for 2 min and 5 % (w/v) chloramine T solution for 5 min; the stem rind was removed and the inner tissues were cut into small pieces with a steril...