Johne’s Disease (JD) is a chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP), primarily affecting a wide variety of agriculturally significant ruminant species such as sheep, cattle, deer and goats. Following ingestion, MAP is translocated across the intestinal epithelium via specialised M cells located in the Peyer’s patches and into the intestinal submucosa whereby MAP localises within host macrophages upon phagocytosis. In recent years, there have been numerous studies that have identified cholesterol as a key factor influencing the establishment of mycobacterial infection within macrophages through prevention of phagocyte maturation. Additionally, cholesterol has been recognised as a major energy source that can be utilised by mycobacterial species. However, despite these implications of cholesterol associated with MAP, the mechanisms driving these cellular alterations are yet to be fully understood. The main objective of this study was to determine if there is a colocalisation of cholesterol with GFP-labelled MAP within macrophages following exposure to MAP. Using fluorescent microscopy, we observed that there was a noticeable redistribution of cholesterol within macrophages exposed to MAP. Furthermore, in macrophages exposed to MAP there was an accumulation of intracellular cholesterol at focal points located throughout the cell. In addition, the expression of cholesterol-associated genes was explored in this study to further understand the relationship between MAP and the host. The findings from this study may represent a mycobacterial adaptation to evade detection during the early stages of infection.