Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of Johne's disease in ruminants, poses challenges to veterinary health and food safety. Despite an immune response that partially controls early infection, Map persists in macrophages through mechanisms not well understood. Here, we explored how the Map major membrane protein (MMP) modulates immune pathways in bovine monocyte-derived macrophages (MoMΦs). MMP is a key component of the bacterial cell membrane recognized in cattle with Johne's disease, making it a critical antigenic target for immune studies. Using high-resolution transcriptomics, we identified that MMP stimulation rapidly activates genes linked to pro-inflammatory cytokine signaling, antigen processing, and presentation via MHC I and II pathways. Gene Ontology and KEGG pathway enrichment analyses highlighted upregulation of TNF, IL-17, and NF-κB signaling cascades, suggesting an immune signaling that may foster cytotoxic T cell development. Phosphorylation assays confirmed that MMP triggers MAPK activation within minutes, implicating both p38 and JNK1/2 in early macrophage responses. Machine learning approaches revealed subtle yet significant MMP-specific gene signatures including ATG5 and ATG12, implicated in autophagosome assembly. These findings point to a dynamic interplay between antibacterial autophagy and immunostimulatory pathways elicited by MMP in bovine macrophages. Importantly, our results suggest the relevance of MMP as a potential vaccine target, as it not only elicits immune-activating signals but also engages host defenses critical to restricting Map survival. Overall, this work provides an ex vivo framework for delineating the molecular underpinnings of Map infection, offering new insights into macrophage-based immunity and informing development of novel therapeutic and prophylactic strategies against paratuberculosis. Our data open avenues for translational studies, illuminating the interplay between MMP, macrophages, and protective host immunity.