Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling.