Main conclusion <p>Transcriptomic and metabolic comparisons reveal putative regulatory and metabolic differences underlying juice sac initiation in citrus fruit.</p> Abstract <p>The edible portion of citrus fruits consists of juice sacs—specialized structures unique among fruits—that develop shortly after anthesis from the endocarp, originating from the innermost layers of the albedo. While their physiological and biochemical properties are well studied, the regulatory mechanisms controlling juice sac initiation remain poorly understood. In this study, we compared two cultivars of citron (<i>Citrus medica</i> L.)—the <i>Calabria</i> citron, which develops juice sacs normally, and the <i>Yemenite</i> citron, which does not—across four developmental stages: closed flowers, flowers at anthesis, and fruitlets at one and two weeks post-anthesis. We performed a comparative transcriptomic analysis of endocarp cells, followed by Weighted Gene Co-Expression Network Analysis (WGCNA) and a metabolomic analysis of whole ovaries and fruitlets. As expected, the <i>Calabria</i> endocarp exhibited higher expression of genes associated with cell wall formation, DNA replication, and cell proliferation, particularly two weeks post-anthesis. In contrast, stress-related genes were more abundant in the <i>Yemenite</i> endocarp. <i>Calabria</i> ovaries and fruitlets showed an increase in amino acids, whereas those of the <i>Yemenite</i> citron exhibited induction of TCA cycle and energy metabolism pathways. Integrating transcriptomic and metabolomic data revealed significant enrichment of carbohydrate and energy metabolism pathways in the <i>Yemenite</i> citron. Additionally, we identified a transcription factor regulatory network that may contribute to juice sac initiation. These findings provide new insights into the molecular processes underlying juice sac initiation and establish a foundation for future research aimed at elucidating its regulatory mechanisms.</p>