Abstract: Gentamycin-resistant gene (GM) was ligated with the flanking fragments of tsh gene amplified by PCR, and then the product was transferred into MCS of pUC18 via DNA recombination techniques. The resulting plasmid was named pUC18-tshFRGM. The fragment of interest was digested and subcloned into suicide vector pMEG375 to generate the plasmid pMEG375- tshFRGM. The plasmid was transformed into APEC E037 used as recipient strain containing tsh gene. ΔTsh-deleted E037 mutant strain was screened by homologous recombination. The animal infection results revealed that the pathogenicity of E037 (Δtsh) strain reduced. Successful construction of Δtsh-deleted E037 mutant strain has an important value and significance for elucidating the function of tsh gene and the role of tsh gene playing in the pathogenicity. The 50% lethal dose (LD50) of E037 and E037 (Δtsh) in commercial day-old chickens experimentally inoculated via intratrachea were 105.6CFU and 109.0CFU, respectively. In the chicken challenge model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. Among 243 avian E.coli isolates, tsh was present in 68.7% (167 strains). 146 (87.4%), 21 (12.6%) and 0 (0%) of 167 tsh+ isolates were high, intermediate and low in pathogenicity, respectively. Among tsh+ isolates, 89.5%-100% of those high pathogenic isolates of O1, O2 or O78 serogroups hold tsh, while 53.3% in other isolates except O1, O2 and O78 serogroups possessed tsh (P<0.01).