FUSCA3 (FUS3) is a member of B3-domain transcription factor family and master regulator of seed development. It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle, especially in seed germination, dormancy, embryo formation, seed and fruit development, and maturation. However, there is limited information about its functions in seed and fruit development of grapevine. In this study, we expressed VvFUS3 in tomato for its functional characterization. Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size. Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected. However, there were no obvious differences in pollen size, shape, and viability, suggesting that VvFUS3 affects seed development but not the pollen grains. Moreover, the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3. These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size. In conclusion, our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.