The influence of solution chemistry and surface macromolecules on cell transport in porous media has been investigated in a well-controlled packed-bed system using Escherichia coli O157:H7. Three different ionic strengths (1, 10, and 100 mM) and two different pHs (8.4 and 9.2) were employed for the experiments. To compliment the transport experiments, the bacterial viability, cell size, hydrophobicity, electrophoretic mobility, acidity (i.e., surface charge density), and extracellular polymeric substances (EPS) composition were characterized. These analyses indicated the cell surface chemistry is not sensitive to solution chemistry; however, the cell transport was considerably influenced by changing solution chemistry. Less retention of bacteria in the column was observed at high ionic strength (1 vs. 100 mM), which cannot be explained by classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. In order to further investigate the role of surface macromolecules of the cells, a proteolytic enzyme (proteinase K) was employed to cleave surface proteins of E. coli O157:H7. The effectiveness of proteinase K was confirmed by FT-IR analysis, indicating that uncharged functional groups have been removed from bacterial surface polymers after enzyme treatment. The results are consistent with complimentary cell characterization results. Specifically, the cells became more negative and hydrophilic than untreated ones. Furthermore, transport behaviors of proteinase K-treated cells will be discussed and compared with the prediction by DLVO theory.