Aims To investigate plasmid-mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 20082010. Methods and Results In total, 2106 faecal specimens from 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents were cultured. The faecal colonization rates of fosfomycin-resistant E.coli were as follows: 11 center dot 2% in pigs, 8 center dot 6% in cattle, 7 center dot 3% in chickens, 2 center dot 4% in dogs, 0 center dot 8% in cats and 1 center dot 5% in rodents. The cultures yielded 1693 isolates of which 831 were extended-spectrum -lactamases (ESBL) producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be producers of ESBL and to have resistance to chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin and tetracycline. Of the 101 fosfomycin-resistant isolates, 97 (96 center dot 0%) isolates were fosA3 positive and 94 (93 center dot 1%) were blaCTX-M positive. PCR mapping showed that the fosA3-containing regions were flanked by IS26, both upstream and downstream in 81 (83 center dot 5%) isolates, and by an upstream blaCTX-M-14-containing transposon-like structure (ISEcp1-blaCTX-M-14-IS903 or ISEcp1-IS10 -blaCTX-M-14-IS903) and a downstream IS26 in 14 (14 center dot 4%) isolates. For the remaining two isolates, fosA3 was flanked by a downstream IS26 but the upstream part cannot be defined. In a random subset of 18 isolates, fosA3 was carried on transferable plasmids with sizes of 50200kb and the following replicons: F2:A-B- (n=3), F16:A1:B- (n=2), F24:A-B- (n=1), N (n=1), B/O (n=1) and untypeable (n=3). Significance and Impact of the Study This study demonstrates the emergence of fosA3-mediated fosfomycin resistance among multidrug-resistant E.coli isolates from various animals. IS26 transposon-like structures might be the main vehicles for dissemination of fosA3.