In this study, we examined sucrose metabolism and expression of invertase, a sucrolytic enzyme, during vegetative and floral development in Eustoma grandiflorum, a widely cultivated ornamental plant. During vegetative growth, sucrose content was relatively lower in roots and unexpanded leaves than in expanded leaves. The activities of cell-wall invertase (CWIN) and vacuolar invertase (VIN) were higher in roots and unexpanded leaves, respectively, whereas the activity of cytoplasmic invertase (CIN) was higher in both organs. During flower development, although the contents of reducing sugars and sucrose were relatively unchanged, starch content was higher in elongated flower buds (stage 2), and we also detected a significant increase in CWIN activity. VIN and CIN showed contrasting changes in enzymatic activity, with the former being higher, and the latter lower in opened flowers (stage 3). Furthermore, we cloned two putative CWIN genes (EgCWIN1 and EgCWIN2), one putative VIN gene (EgVIN1), and one putative CIN gene (EgCIN1), and examined the transcript levels of these four genes. Although we detected no clear correlations between invertase activities and the transcript levels of invertase genes in vegetative organs, we observed changes in the transcript levels of EgCWIN1, EgVIN1, and EgCIN1 corresponding to changes in activities of the respective invertase during flower development. These results indicate that carbon partitioning during vegetative and floral development in E. grandiflorum is controlled by three invertase isoforms, and that differential gene expression underlies the successive induction of these invertase isoforms during flower opening.