Infection of mammary gland cells with bacterial pathogens begins with adhesion, invasion, and persistence within the cells or systemic distribution. Some bacteria, such as Escherichia coli, are known to causes bovine mastitis, resulting in acute proinflammatory responses in the mammary tissue. Mycobacterium avium ssp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is able to spread to distant organs after crossing intestinal cells, reaching the mammary gland and potentially being released in milk, infecting calves during suckling. Its exit from systemic sites may be influenced by preexisting inflammation such as that caused by E. coli mastitis. Interactions between E. coli and MAP in mammary epithelial cells have not yet been described. In this study, we posited that E. coli-infected bovine mammary epithelial cells would facilitate baso-apical translocation of MAP in an ex vivo model. We showed that the presence of E. coli in a bovine mammary epithelial cell line (MAC-T) increased baso-apical translocation of MAP to the apical side of the cells. Levels were significantly higher 30 min post-infection and decreased at 120 min post-infection. Cells previously infected with E. coli and MAP or with E. coli alone showed a significant increase in IL1B mRNA expression at 120 min. We detected no significant expression of p38 mitogen-activated protein kinase (mapkp38) or IL10, regardless of treatment. Thereby, the presence of E. coli in MAC-T cells alters the translocation of MAP through epithelial cells, enabling its rapid translocation to the cellular surface. Expression of IL1B was shown to influence the apical-basal translocation of MAP at 120 min. Findings from the current study suggest that MAP translocation into milk is likely enhanced by inflammatory states such as those induced during E. coli mastitis. This is the first report demonstrating the effect of E. coli under MAP coinfection in bovine mammary epithelial cells under experimental conditions.