Objective: Obesity hypoventilation syndrome is associated with diaphragmatic dysfunction. This study aimed to explore the role of endoplasmic reticulum (ER) stress in mediating obesity-induced diaphragmatic dysfunction.Methods: A pulmonary function test and ultrasound were applied to evaluate diaphragmatic function and magnetic resonance imaging was applied to measure diaphragmatic lipid deposition in human patients. For the mechanistic study, obese mice were introduced to a high-fat diet for 24 weeks, followed by diaphragmatic ultrasound measurement, transcriptomic sequencing, and respective biochemical analysis. Automatic force mapping was applied to measure the mechanical properties of C2C12 myotubes.Results: People with obesity showed significant diaphragm weakness and lipid accumulation, which was further confirmed in obese mice. Consistently, diaphragms from obese mice showed altered gene expression profile in lipid metabolism and activation of ER stress response, indicated by elevated protein kinase R-like ER kinase (PERK) Xiaoxin Xiang, Yanhua Zhu, and Xuya Pan contributed equally to this study.Xiaoyue Zhang, Yanming Chen, and Guojun Shi are also equal contributors.