BACKGROUND: Macrophage-mediated excessive inflammatory responses play a pivotal role in sepsis progression, particularly in the lungs. Endoplasmic reticulum stress (ERS) and mitochondrial dysfunction, also contribute to the development of acute lung injury (ALI). Dendrobine is a natural alkaloid known to alleviate ERS in various diseases; however, its specific role in sepsis-associated ALI is not yet fully understood. OBJECTIVE: This study investigates the protective effects of dendrobine against ALI and elucidates the underlying mechanisms. METHODS: We assessed the protective effects of dendrobine using LPS-induced THP-1 cells and a murine model of sepsis. Techniques included HE staining for lung tissue damage, TUNEL staining for pulmonary apoptosis, flow cytometry for intracellular reactive oxygen species (ROS) levels and apoptosis, transmission electron microscopy for ER autophagy and changes in mitochondria-associated ER membranes (MAMs), and confocal microscopy for ER autophagy flux, mitochondrial membrane potential, and mitochondrial calcium ion levels. RESULTS: Dendrobine significantly alleviated LPS-induced ALI and inflammatory responses in mice by reducing the expression of ERS-related and pro-apoptotic proteins. It enhanced ER autophagy by upregulating FAM134B, inhibiting its ubiquitination and degradation, and promoting its interaction with LC3B. Dendrobine partially restored LPS-induced mitochondrial dysfunction by reducing MAM formation. Conversely, FAM134B knockdown reversed the protective effects of dendrobine. CONCLUSION: This study demonstrates that dendrobine alleviates LPS-induced ERS and mitochondrial dysfunction through FAM134B-mediated ER autophagy and MAM reduction, thereby protecting lung tissue. These findings highlight the potential of dendrobine as a therapeutic agent against sepsis-induced ALI.