Alterations in the outer membrane (OM) proteome in response to tetracycline have been reported in Escherichia coli K-12, but information regarding their functional profile is not available. In this study, upregulation of FimD, Tsx, OmpW, OmpC and TolC and downregulation of LamB in response to tetracycline were detected and confirmed by two-dimensional gel electrophoresis subproteomics and Western blotting, respectively. The ability of the six altered OM proteins was investigated using genetically modified strains with gene deletion or complementation. Of the six mutants tested, DeltatolC and DeltaompC showed decreased minimum inhibitory concentrations (MICs) and DeltalamB showed an increased MIC. These changes in the three mutants could be completely recovered by gene complementation. The survival capability of gene-deleted was significantly increased in DeltalamB mutants and significantly decreased in DeltatolC, DeltaompC, DeltaompW and Deltatsx with respect to the control; a more significant decrease was observed in DeltatolC and DeltaompC than DeltaompW and Deltatsx. These changes were also observed in their complemented strains. Our results indicate that LamB, OmpC and TolC are important OM proteins for tetracycline resistance in E. coli. Meanwhile, our data suggest that functional validation is required to investigate whether the altered proteins obtained from two-dimensional gel electrophoresis play a direct or indirect role in phenotypic changes.