Speculation about the association of soil characteristics with the expression of ovine Johne's disease (OJD) prompted this cross-sectional study. We enrolled 92 sheep flocks in Australia during 2004-2005 and in each enrolled flock collected pooled faecal samples from an identified cohort (group of same age and sex) of sheep and soil samples from the paddocks grazed by this cohort of sheep. Faecal pools were cultured to create three outcome variables: positive or negative status of faecal pools (pool OJD status, binary); the log number of viable Mycobacterium avium subsp. paratuberculosis (MAP) organisms per gram of faeces (log pool MAP number, continuous); and the prevalence of faecal shedders (cohort OJD prevalence level, ordinal: low <2%, medium 2-10% and high >10%). Separate statistical models were then developed to investigate the association between soil characteristics and each outcome variable. Sheep raised on soils with a higher percentage of organic carbon and clay had a higher OJD prevalence whereas, sheep grazing on soils with a higher content of sand and nitrogen had a lower OJD prevalence. Iron content of the soil was positively associated with OJD infection but the association between soil pH and OJD was inconclusive. Parent soil type, the only farm level factor, was not significant in any of the final models. Study results indicate a higher risk of OJD in sheep raised on soils with greater organic matter and clay content. We hypothesise that this is due to adsorption of MAP to clay and the consequent retention of the bacteria in the topsoil, thus making them available in higher numbers to grazing sheep.