To study in vivo the role of the ubiquitin-like protein Smt3 (Sumo) during Drosophila development, we generated transgenic flies carrying the transgene UAS-smt3i to reduce smt3 mRNA levels in specific groups of cells. Low smt3 in the prothoracic gland, the tissue responsible for the synthesis of ecdysteroids, prevents metamorphosis. RNAi knockdown larvae stop their development in their last larval stage and remain alive for up to a month, during which they continue to eat and gain weight. Their prothoracic glands have fewer, but larger cells than normal, similar to larvae mutant in lesswright, the homologue of the Sumo conjugating enzyme gene Ubc9. They also have lower ecdysteroid titre than WT. After dietary administration of exogenous ecdysone some of these larvae form pupal cases, but do not proceed further in development and die. However, addition of cholesterol or 7-dehydrocholesterol does not rescue the larval phenotype, indicating that sumoylation must be necessary for later steps in the ecdysteroid synthesis pathway. Interestingly, we observed that, in larvae with lower levels of smt3, as well as in lesswright mutants, the subcellular localization and expression levels of factors involved in the regulation of ecdysteroids synthesis, are altered, including Molting defective, Without children, _-Ftz transcription factor 1 and Disembodied. We also investigate the sumoylation capacity of these and other factors involved in ecdysteroid synthesis.