The isolated middle tobacco leaf of flue–cured tobacco variety Qinyan96 was used as the material and three water loss treatments (CK with no water loss, T1 with 10% water loss and T2 with 20% water loss) were set up in the experiment to study the respiratory rate and the activities and gene expression of starch synthesis related enzymes as ADP–glucose pyrophosphorylase (AGP) and Granule bound starch synthetase (GBSS) in post harvest tobacco leaves. The results showed that compared with CK, the breathing peak of post harvest tobacco leaves with 10% and 20% dehydration happened respectively 6 h and 12 h earlier, the respiration rate increased by 20% and 8% respectively, and the starch content reduced by 30% and 14% respectively. Under dehydration condition, activities of AGP and GBSS enzymes in tobacco leaves decreased rapidly in the former period (12 h) after harvest which promoted a rapid decline in enzyme activity in the later period. Further gene expression analysis showed that the expression levels of AGP and GBSS genes in post harvest tobacco leaves with dehydration first increased and then decreased with prolonged post harvest time, indicating dehydration inhibited starch anabolism and promoted rapid degradation of starch in the later period (above 12 h).