Magnesium (Mg) is an essential macronutrient supporting various functions, including photosynthesis. However, the specific physiological responses to Mg deficiency remain elusive. In this study, 2-week-old rice seedlings (Oryza sativa. cv. Nipponbare) with three expanded leaves (L2–L4) were transferred to Mg-free nutrient solution for 8 days. In the absence of Mg, on day 8, L5 and L6 were completely developed, while L7 just emerged. We also studied several mineral deficiencies to identify specific responses to Mg deficiency. Each leaf was analyzed in terms of chlorophyll, starch, anthocyanin and carbohydrate metabolites, and only absence of Mg was found to cause irreversible senescence of L5. Resupply of Mg at various time points confirmed that the borderline of L5 death was between days 6 and 7 of Mg deficiency treatment. Decrease in chlorophyll concentration and starch accumulation occurred simultaneously in L5 and L6 blades on day 8. However, nutrient transport drastically decreased in L5 as early as day 6. These data suggest that the predominant response to Mg deficiency is a defect in transpiration flow. Furthermore, changes in myo-inositol and citrate concentrations were detected only in L5 when transpiration decreased, suggesting that they may constitute new biological markers of Mg deficiency.