Background <p>The Notch signaling pathway plays a critical role in maintaining immune responses. This study examined the regulatory effect of the Notch signaling pathway on mitophagy and macrophage function in response to <i>Mycobacterium bovis</i> Bacillus Calmette-Guérin (BCG) infection.</p> Methods <p>RAW264.7 cells with stable Notch-1 knockdown were divided into three groups: control, siRNA Notch-1, and siRNA Notch-1 + valinomycin. Following BCG infection, we measured reactive oxygen species (ROS) levels, the expression of Notch signaling and mitophagy-related genes, cytokines, and BCG colony count.</p> Results <p>RAW264.7 cells infected with 1919-siRNA lentivirus exhibited significantly decreased expression of Notch-1, indicating stable Notch-1 knockdown. Following BCG infection, the expression of Notch-1 decreased in the siRNA Notch-1 group and the siRNA Notch-1 + valinomycin group compared to the control group. Moreover, the expression levels of JAG1, autophagy markers (Beclin 1 and LC3), and mitophagy markers (Drp-1 and Nix) were significantly increased in the siRNA Notch-1 + valinomycin group. Additionally, the ROS level showed a significant increase in the siRNA Notch-1 group, which was further augmented after treatment with mitophagy inducer valinomycin. The BCG clearance rate decreased in the siRNA Notch-1 group but increased after treatment with valinomycin. IL-6 levels were significantly elevated in the siRNA Notch-1 group and further increased in the siRNA Notch-1 + valinomycin group. However, TNF-α levels significantly increased in the siRNA Notch-1 group and decreased significantly in the siRNA Notch-1 + valinomycin group.</p> Conclusion <p>The Notch signaling pathway inhibits mitophagy and macrophage-mediated elimination of BCG.</p>