AbstractSeedless grapes are increasingly popular throughout the world, and the development of seedless varieties is a major breeding goal. In this study, we demonstrate an essential role for the grapevine MADS-box geneVvMADS28in morphogenesis of the ovule. We found thatVvMADS28mRNA accumulated in the ovules of a seeded cultivar, ‘Red Globe’, throughout the course of ovule and seed development, especially within the integument/seed coat. In contrast, in the seedless cultivar ‘Thompson Seedless’,VvMADS28was expressed only weakly in ovules, and this was associated with increased levels of histone H3 lysine 27 trimethylation (H3K27me3) within theVvMADS28promoter region.RNAi-mediated transient suppression ofVvMADS28expression in ‘Red Globe’ led to reduced seed size associated with inhibition of episperm and endosperm cell development. Heterologous overexpression ofVvMADS28in transgenic tomatoes interfered with sepal development and resulted in smaller fruit but did not obviously affect seed size. Assays in yeast cells showed thatVvMADS28is subject to regulation by the transcription factorVvERF98, and that VvMADS28 could interact with the Type I/ Mβ MADS-domain protein VvMADS5. Moreover, through DNA-affinity purification-sequencing (DAP-seq), we found that VvMADS28 protein specifically binds to the promoter of the grapevineWUSCHEL(VvWUS) gene, suggesting that maintenance of the VvMADS28–VvMADS5 dimer andVvWUSexpression homeostasis influences seed development. Taken together, our results provide insight into regulatory mechanisms of ovule and seed development associated withVvMADS28.