Aims: This study sought to analyze the molecular mechanisms contributing to the development of rifaximin (Rfx) resistance in vitro in Escherichia coli. Twenty-eight Rfx-resistant mutants as well as four clinical isolates of E. coli were analyzed. The results obtained show that mutations in the rpoB gene and overexpression of Phe-Arg-β-naphthylamide (PAβN)-inhibitible efflux pump were implicated in Rfx resistance. Results: Amino acid substitutions at position 516 of the β-subunit of RNA polymerases were the most frequently obtained (53.6% of the mutants). The efflux pump inhibitor decreased the minimal inhibitory concentration (MIC) of 71.43% (20/28) of the mutant strains. Conclusions: Mutations studied in the rpoB gene and overexpression of PAβN-inhibitible efflux pumps contribute to Rfx resistance (together or not), whereas alterations in porin levels do not seem to have a relevant role in the acquisition of Rfx resistance. © 2012 Mary Ann Liebert, Inc.