Aberrant lipid metabolism disrupts calcium homeostasis and causes liver endoplasmic reticulum (ER) stress in obesity. This study investigates the mechanisms underlying chronic ER stress in obesity by comparing the proteomic and lipidomic profiles of hepatic ER in lean and obese mice. The results show that in obese mice, lipid synthesis is increased while protein synthesis is suppressed, leading to a shift in ER function from protein to lipid metabolism. This shift results in altered ER lipid composition, which inhibits SERCA (sarco/endoplasmic reticulum calcium ATPase) activity and causes ER stress. Correcting the ER lipid composition or overexpressing SERCA in obese mice reduces ER stress and improves glucose homeostasis. The study also found that the PC/PE ratio in the ER is elevated in obese mice, which impairs SERCA function and contributes to ER stress. Reducing Pemt expression, which is involved in PC synthesis, lowers the PC/PE ratio and improves calcium transport, reducing ER stress and improving glucose metabolism. Overexpression of SERCA in obese mice also alleviates ER stress and improves glucose homeostasis. The findings suggest that lipid metabolism plays a critical role in ER stress and calcium homeostasis in obesity. Abnormal lipid metabolism leads to impaired ER function, which contributes to the development of insulin resistance and diabetes. The study highlights the importance of maintaining proper lipid and calcium metabolism to prevent ER stress and its associated metabolic disorders. The results provide a framework for understanding the pathogenesis of hepatic lipid metabolism and chronic ER stress in obesity, and suggest that interventions targeting hepatic phospholipid synthesis and ER calcium homeostasis could be promising therapeutic strategies for obesity-related diseases.Aberrant lipid metabolism disrupts calcium homeostasis and causes liver endoplasmic reticulum (ER) stress in obesity. This study investigates the mechanisms underlying chronic ER stress in obesity by comparing the proteomic and lipidomic profiles of hepatic ER in lean and obese mice. The results show that in obese mice, lipid synthesis is increased while protein synthesis is suppressed, leading to a shift in ER function from protein to lipid metabolism. This shift results in altered ER lipid composition, which inhibits SERCA (sarco/endoplasmic reticulum calcium ATPase) activity and causes ER stress. Correcting the ER lipid composition or overexpressing SERCA in obese mice reduces ER stress and improves glucose homeostasis. The study also found that the PC/PE ratio in the ER is elevated in obese mice, which impairs SERCA function and contributes to ER stress. Reducing Pemt expression, which is involved in PC synthesis, lowers the PC/PE ratio and improves calcium transport, reducing ER stress and improving glucose metabolism. Overexpression of SERCA in obese mice also alleviates ER stress and improves glucose homeostasis. The findings suggest that lipid metabolism plays a critical role in ER stress and calcium homeostasis in obesity. Abnormal lipid metabolism leads to impaired ER function, which contributes to the development of insulin resistance and diabetes. The study highlights the importance of maintaining proper lipid and calcium metabolism to prevent ER stress and its associated metabolic disorders. The results provide a framework for understanding the pathogenesis of hepatic lipid metabolism and chronic ER stress in obesity, and suggest that interventions targeting hepatic phospholipid synthesis and ER calcium homeostasis could be promising therapeutic strategies for obesity-related diseases.