The causes of reproductive failure under drought stress (DS) are poorly understood. We hypothesized that reproductive failure was related to drought-induced changes in pistil biochemistry. To address this hypothesis, a water deficit-induced experiment was conducted with two cotton cultivars (Dexiamian 1, drought tolerant; Yuzaomian 9110, drought sensitive). Results showed that DS decreased the photosynthesis of subtending leaf and downregulated sucrose transporter gene (GhSUT-1) expression in pistil for both cultivars, resulting in lower pistil carbon accumulation which was reflected in the decreased starch accumulation. Lower starch, as potential energy, and adenosine triphosphate (ATP), as direct energy, in droughted pistils suggested less energy for pollen tube entrance into ovules, reducing the fertilized ovule number and fertilization efficiency. Further, although pistil peroxidase activity increased under DS, a higher hydrogen peroxide (H 2 O 2 ) level still was measured in droughted pistils than well-watered pistils, damaging reproductive activities. Moreover, larger decreases in photosynthesis, pistil GhSUT-1 expression, carbon accumulation, starch and ATP contents caused by DS for Yuzaomian 9110 than Dexiamian 1, and different responses of superoxide dismutase and catalase activities, and ascorbic acid and H 2 O 2 contents to DS between the two cultivars might be the reasons causing a greater decrease in fertilization efficiency for Yuzaomian 9110 than Dexiamian 1 under DS. Thus, we suggest that decreased ovule fertilization under DS was related to the disorganized carbohydrate metabolism and inefficient antioxidant defense in droughted pistils, and the effects of DS on pistil carbohydrate metabolism and antioxidant defense were more significant for drought-sensitive cultivars than drought-tolerant cultivars.