Gram-negative bacterial infections pose a significant threat to public health. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and induces innate immune responses, autophagy, and cell death, which have major impacts on the body’s physiological homeostasis. However, the role of TLR4 in bacterial LPS-induced autophagy and apoptosis in large mammals, which are closer to humans than rodents in many physiological characteristics, remains unknown. So far, few reports focus on the relationship between TLR, autophagy, and apoptosis in large mammal levels, and we urgently need more tools to further explore their crosstalk. Here, we generated a TLR4-enriched mammal model (sheep) and found that a high-dose LPS treatment blocked autophagic degradation and caused strong innate immune responses and severe apoptosis in monocytes/macrophages of transgenic offspring. Excessive accumulation of autophagosomes/autolysosomes might contribute to LPS-induced apoptosis in monocytes/macrophages of transgenic animals. Further study demonstrated that inhibiting TLR4 downstream NF-κB or p38 MAPK signaling pathways reversed the LPS-induced autophagy activity and apoptosis. These results indicate that the elevated TLR4 aggravates LPS-induced monocytes/macrophages apoptosis by leading to lysosomal dysfunction and impaired autophagic flux, which is associated with TLR4 downstream NF-κB and MAPK signaling pathways. This study provides a novel TLR4-enriched mammal model to study its potential effects on autophagy activity, inflammation, oxidative stress, and cell death. These findings also enrich the biological functions of TLR4 and provide powerful evidence for bacterial infection.