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Expression of thin aggregative fimbriae (Agf) in Salmonella, which is responsible for bacterial cell adhesion to surfaces, aggregation, and formation of biofilms, is regulated by a complex mechanism. In order to identify gene(s) involved in the expression of Agf, the TnphoA transposon was introduced into Salmonella typhimurium χ8505 for random mutagenesis. Colonies showing a change from wrinkly-rough morphology to the smooth form were screened for candidates. Through multiple selection processes, a mutant, named S. typhimurium CK167 was selected as the final candidate. Analyses of the nucleotide sequences of TnphoA insertion site identified the insertion in rpoE gene. S. typhimurium CK178, a defined rpoE deletion mutant on χ8505, exhibited the same colony morphology as seen in CK167. The S. typhimurium CK178 strain expressed significantly reduced amounts of AgfD and showed modulated biofilm formation, demonstrating the role of RpoE in AgfD expression. To the best of our knowledge, this is the first report demonstrating that RpoE acts as a regulator in the expression of Agf in Salmonella.