Mycobacterium avium subspecies paratuberculosis (MAP) and Mycobacterium avium subspecies avium (MAA) represent two closely related intracellular bacteria with vastly different associated pathologies. MAA can cause severe respiratory infections in immune compromised humans but is nonpathogenic in ruminants and is more readily controlled by the bovine immune system than MAP. MAP causes a fatal wasting syndrome in ruminants, typified by granulomatous enteritis localized in the small intestine. MAP has also been cited as a potential cause of human Crohn's disease. We used a bovine immune-specific microarray (BOTL-5) to compare the response of mature bovine monocyte-derived macrophages (MDM cells) to MAP and MAA. Statistical analysis of microarray data revealed 21 genes not appreciably expressed in resting MDM cells that were activated following infection with either MAA or MAP. Further analysis revealed 144 genes differentially expressed in MDM cells following infection with MAA and 99 genes differentially expressed following infection with MAP. Of these genes, 37 were affected by both types of mycobacteria, with three being affected in opposite directions. Over 41% of the differentially expressed genes in MAA and MAP infected MDM cells were members of, regulated by, or regulators of the MAPK pathways. Expression of selected genes was validated by quantitative real-time reverse transcriptase PCR and in several key genes (i.e., IL-2 receptor, tissue inhibitor of matrix metalloproteinases-1, and Fas-ligand) MAA was found to be a stronger activating factor than MAP. These gene expression patterns were correlated with prolonged activation of p38 MAPK and ERK1/2 by MAA, relative to MAP.