Technical Abstract: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection causing diarrhea and wasting in cattle, sheep, and other ruminants. Because Johne’s disease is spread by the ingestion of M. paratuberculosis, a good understanding of this organism’s transport behavior is needed to minimize the transmission of this disease, yet surprisingly little is known about this organism’s ability to move through the environment. To begin to fill in this gap in our knowledge, we investigated the processes controlling the transport of M. paratuberculosis through aquifer materials. In particular, we measured important surface characteristics known to affect bacterial attachment to sediment surfaces, namely surface charge and hydrophobicity, and conducted transport experiments through clean and Fe-coated sands. We found that M. paratuberculosis has a strong negative charge and is highly hydrophobic. We observed minimal transport of M. paratuberculosis through short laboratory columns suggesting that the potential for groundwater contamination by M. paratuberculosis is low; however, the organism may remain bound to the soil near the surface where it can be ingested by grazing animals or be released during run off events. This research provides important information for understanding the movement of M. paratuberculosis in the environment.