Hydration-dehydration cycles can frequently cause stress to seeds, but can also be used to improve germination. However, the molecular basis of the stress caused is poorly understood. Herein, we examine the effects of hydration-dehydration cycles on seed viability and profile the membrane glycerolipid molecular species. We find that seed viability was not affected during the first two cycles, but significantly decreased as further cycles were applied, until all viability was lost. The abundances of seven glycerolipid classes increased and decreased through hydration and dehydration, respectively, but the phosphatidic acid and diacylglycerol abundances changed in the opposite sense, while total glycerolipid contents remained constant. This suggests that during hydration-dehydration cycles, turnover of glycerolipid metabolite pools take place, while no significant lipid synthesis or degradation is involved. As further hydration-dehydration cycles occurred, lipid unsaturation increased, plastidic lipids decreased, and phosphatidylserine acyl chains lengthened. The latter two could be lethal for seeds. Our findings reveal a novel model of membrane lipid changes, and provide new insights into the responses of seeds to hydration-dehydration cycles.