Mongolian gerbils (Meriones unguiculatus) have high physiological flexibility in response to acute temperature changes, and have the widest thermoneutral zone (TNZ, 26.5-38.9 °C) reported among small mammals. At the upper critical temperature (T(uc), 38.9 °C), body temperatures of gerbils were significantly increased (39-41 °C) while metabolic rates were maintained at the basal level. In contrast, below the lower critical temperature (T(lc), 26.5 °C), metabolism was elevated and body temperature stable. Rapid changes in mitochondrial membrane lipidome were hypothesized to play an important role during acute thermoregulation of gerbils. Taking advantage of a recent lipidomic technique, we examined changes in the membrane phospholipids environment and free fatty acids (FFA) production in mitochondria between 38 and 27 °C (in the TNZ), and between 27 and 16 °C (below the TNZ). At 38 °C, acute heat stress elicited distinct remodeling in mitochondrial membrane lipidome which related to a potential decrease in mitochondrial respiration and membrane fluidity compared to 27 °C. At 16 °C, a sharply decreased unsaturation index and increased chain lengths were detected in mitochondrial FFA production both in muscle and brown adipose tissue. Our results suggest that mitochondrial membrane lipid remodeling may stabilize membrane function and activity of respiration related membrane protein to maintain a stable metabolic rate at T(uc), and improve heat production by decomposing less fluid fatty acid conjugates of membrane lipids under acute cold exposure. These data therefore imply an important role of membrane remodeling during acute thermoregulation in a non-hibernating endotherm.