Background The emergence of fosfomycin resistance and extended-spectrum β-lactamase (ESBL) genes is a serious threat to public health and a new challenge in shigellosis treatment. The purpose of this study was to identify fosfomycin resistance and characterize β-lactamase genes in fos-carrying isolates of Shigella flexneri from patients in China. Methods A total of 263 S. flexneri isolates were collected from 34 hospitals in the Anhui Province of China during September 2012-September 2015 and screened for fosA3 , fosA , and fosC2 by PCR amplification and sequencing. The fos-carrying isolates were then screened for β-lactamase genes. The clonal relationships between fosA3 -carrying isolates, the transmissibility of fosfomycin resistance, replicon types of plasmids carrying fosfomycin resistance genes and other associated resistance genes were investigated. Results Twenty-five of the 263 isolates (9.5%) showed resistance to fosfomycin, and 18 (6.8%) were positive for fosA3 . None of the isolates was positive for fosA or fosC2 . Seventeen of the isolates carrying fosA3 (94%) were CTX-M producers (seven CTX-M-55, five CTX-M-14, and five CTX-M-123), while three (16.7%) were TEM producers (TEM-1).Sixteen (88.9%) fosA3 -carrying isolates exhibited multi-drug resistance. The replicon types of the 13 fosA3 -carrying plasmids were IncF (n=13), IncHI2 (n=3), IncIl-Ir (n=2), and IncN (n=1). Conclusions Our results indicated that fosA3 could spread through plasmids in S. flexneri isolates, along with the bla CTX-M and bla TEM , which facilitate its quick dispersal. To the best of our knowledge, this is the first report of CTX-M-123-type ESBLs in S. flexneri isolates from patients in China.