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ABSTRACTExtraintestinal pathogenic Escherichia coli(ExPEC) is an important zoonotic pathogen. Recently, ExPEC has been reported to be an emerging problem in pig farming. However, the mechanism of pathogenicity of porcine ExPEC remains to be revealed. In this study, we constructed a transposon (Tn) mutagenesis library covering Tn insertion in over 72% of the chromosome-encoded genes of a virulent and multi-drug resistant porcine ExPEC strain PCN033. By using a mouse infection model, a transposon-directed insertion site sequencing (TraDIS) assay was performed to identify in vivofitness factors. By comparing the Tn insertion frequencies between the input Tn library and the recovered library from different organs, 64 genes were identified to be involved in fitness during systemic infection. 15 genes were selected and individual gene deletion mutants were constructed. The in vivofitness was evaluated by using a competitive infection assay. Among them, ΔfimGwas significantly outcompeted by the WT strain in vivoand showed defective adhesion to host cells. rfawhich was involved in lipopolysaccharide biosynthesis was shown to be critical for in vivofitness which may have resulted from its role in the resistance to serum killing. In addition, several metabolic genes including fepB, sdhC, fepG, gltS, dcuA, ccmH, ddpD, narU, glpD, malM, and yabLand two regulatory genes metJand baeSwere shown as important determinants of in vivofitness of porcine ExPEC. Collectively, this study performed a genome-wide screening for in vivofitness factors which will be important for understanding the pathogenicity of porcine ExPEC.