Laboratory and numerical studies were conducted to investigate the transport and release of Escherichia coli D21g in preferential flow systems with artificial macropores under different ionic strength (IS) conditions. Macropores were created by embedding coarse sand lenses in a fine sand matrix and altering the length, continuity, and vertical position of the lens. The length of an artificial macropore proved to have a great impact on the preferential transport of E. coli D21g, especially under high-IS conditions. A discontinuous macropore (interrupted by fine sand) was found to have less preferential transport of E. coli D21g than a continuous macropore of the same length that was open to either the top or bottom boundary. At low IS, more extensive transport in the preferential path and earlier arrival time were observed for E. coli D21g than Br− as a result of size exclusion. Two release pulses (one from the preferential path and the other from the matrix) were observed following a reduction of the solution IS for flow systems with macropores that were open to either the top or bottom boundary, whereas three pulses (two from the preferential path and another from the matrix) were observed for systems with discontinuous macropores. Numerical simulations of E. coli D21g under both constant and transient solution chemistry conditions had very high agreement with the experiment data, except for their capability to predict some subtle differences in transport between the various lens configurations.