Johne’s disease is a chronic granulomatous enteritis affecting significant production species such as cattle, sheep and goats. This disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) is associated with extended periods of bacterial dormancy prior to development of clinical signs. In other mycobacterial diseases cholesterol plays a vital role in the establishment of infection and persistence within host cells and Mycobacterium tuberculosis is capable of catabolising cholesterol as a primary energy source. However, there is a lack of knowledge of the relationship between cholesterol and MAP infection. The aim of this study was to examine the role of serum and intracellular cholesterol during the early stages of MAP infection in both sheep and cattle. Using a well-established infection model, sheep and cattle were exposed to MAP and blood and faecal samples were collected monthly from 10 controls and 20 MAP exposed animals (for both sheep and cattle). Serum cholesterol was measured using a colorimetric assay. Monocytes were isolated from control animals, exposed to GFP-tagged MAP in vitro and changes in the localisation of intracellular cholesterol were monitored using immunofluorescence microscopy. Additionally, qPCR was used to analyse changes in the expression of cholesterol-associated genes. Total serum cholesterol changed significantly in exposed animals during the first few months of infection in both sheep and cattle when compared to control animals. In vitro experiments demonstrated that MAP co-localised to cholesterol-rich domains within cells and the expression of cholesterol metabolism associated genes was altered in infected cells. This study has demonstrated that MAP infection may result in the modulation of intracellular cholesterol pathways during the early stages of infection which may be important for the pathogenicity of this organism.