Multidrug-resistant Salmonella Enteritidis (S. Enteritidis) isolates have become a significant threat to public health, and fosfomycin has been proposed as one of the therapeutic antibiotics for serious infections by resistant pathogens. In this study, a total of 501 clinical S. Enteritidis isolates were screened and 14 (2.8%) isolates exhibited resistance to fosfomycin (MIC ≥ 1,024 μg/mL) as well as ceftriaxone (MIC ≥ 128 μg/mL). The fosA3 gene was identified in these 14 isolates. The fosA3 gene that co-transferred with bla was observed on the IncFII plasmids with sizes of ~ 78 (n = 7) or ~ 111 (n = 2) kbp in 9 transconjugants. The fosA3-bearing plasmid p12367A is 111,764 bp in length and possessed a typical IncFII backbone. A 7.6-kbp multidrug resistance region (MRR) was identified in p12367A, which was comprised of fosA3 and bla genes interspersed with ΔISEcp1 and three copies of IS26. Two typical antibiotic resistance determinants (IS26-orf3-orf2-orf1-fosA3-IS26 and IS26-orf477-bla -ΔISEcp1-IS26) shared one IS26 in the MRR. The genetic arrangement of the MRR may have resulted from the stepwise integration of IS26 mobile elements via homologous recombination. Horizontal transfer of IncFII plasmids might contribute to the dissemination of fosA3 and bla resistance genes in S. Enteritidis interspecies. These findings underline further challenges for the prevention and treatment of Enterobacteriaceae infections posed by epidemic IncFII plasmids bearing fosA3-bla .