AGAMOUS like-11 (AGL11) is a D-class MADS-box gene that determines ovule identity in model species. In grapevine, VviAGL11 has been proposed as the main candidate gene responsible for seedlessness because ovules develop into seeds after fertilization. Here, we demonstrate that AGL11 has a direct role in the determination of the seedless phenotype. In grapevine, broad expression analysis revealed very low expression levels of the seedless allele compared to the seeded allele at the pea-size berry stage. Heterozygous genotypes have lower transcript accumulation than expected considering the diploid nature of grapevine, thereby revealing that the dominant phenotype previously described for seedlessness is based on its expression level. In a seeded somatic variant of Sultanina (Thompson Seedless) that has well-developed seeds, Sultanine Monococco, structural differences were identified in the regulatory region of VviAGL11. These differences affect transcript accumulation levels and explain the phenotypic differences between the two varieties. Functional experiments in tomato demonstrated that SlyAGL11 gene silencing produces seedless fruits and that the degree of seed development is proportional to transcript accumulation levels. Furthermore, the genes involved in seed coat development, SlyVPE1 and SlyVPE2 in tomato and VviVPE in grapevine, that are putatively controlled by SlyAGL11 and VviAGL11, respectively, are expressed at lower levels in silenced tomato lines and in seedless grapevine genotypes. In conclusion, this work provides evidence that the D-class MADS-box AGL11 plays a major and direct role in seed development in fleshy fruits, providing a valuable tool for further analysis of fruit development.