Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide.Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection.In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake.TonB family protein (TbfA) was not investigated, since it was failed to knockout at that time.Here, we used a plasmid based gene overexpression and knockdown to investigate it.First, we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters.The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5× 10-5 antibiotic-resistant transconjugants per recipient cell.Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established.Knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organisms growth ability in TSB medium but did not affect its hemin utilization.In contrast, over-expression of TbfA in Riemerella anatipestifer ATCC11845△tonBl△tonB2 significantly promoted the organisms growth in TSB medium but significantly inhibited its hemin utilization.Collectively, these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer.