AbstractRNA interference is a eukaryotic regulatory mechanism by which small non‐coding RNAs typically mediate specific silencing of their cognate genes. In Drosophila, the RNase III enzyme Dicer‐2 (Dcr‐2) is essential for biogenesis of endogenous small interfering RNAs (endo‐siRNAs), which have been implicated in regulation of endogenous protein‐coding genes. Although much is known about microRNA‐based regulatory networks, the biological functions of endo‐siRNAs in animals remain poorly understood. We performed gene expression profiling on Drosophila dcr‐2 null mutant pupae to investigate transcriptional effects caused by a severe defect in endo‐siRNA production, and found 306 up‐regulated and 357 down‐regulated genes with at least a twofold change in expression compared with the wild type. Most of these up‐regulated and down‐regulated genes were associated with energy metabolism and development, respectively. Importantly, mRNA sequences of 39% of the up‐regulated genes were perfectly complementary to the sequences of previously reported endo‐siRNAs, suggesting they may be direct targets of endo‐siRNAs. We confirmed up‐regulation of five selected genes matching endo‐siRNAs and concomitant down‐regulation of the corresponding endo‐siRNAs in dcr‐2 mutant pupae. Most of the potential endo‐siRNA target genes were associated with energy metabolism, including the citric acid cycle and oxidative phosphorylation in mitochondria, implying that these are major metabolic processes directly affected by endo‐siRNAs in Drosophila. Consistent with this finding, dcr‐2 null mutant pupae had lower ATP content compared with controls, indicating that mitochondrial energy production is impaired in these mutants. Our data support a potential role for the endo‐siRNA pathway in energy homeostasis through regulation of mitochondrial metabolism. J. Cell. Biochem. 114: 418–427, 2013. © 2012 Wiley Periodicals, Inc.