SummaryThe whitefly (Bemisia tabaci), a notorious insect vector, transmits hundreds of viruses causing serious yield losses in a diverse food and fiber crops including beans, cassava, cotton, cucurbits, pepper, sweet potato and tomato, and results in billions of U.S. dollars of economic losses annually worldwide. To investigate the molecular mechanisms regulating gene expression in whitefly that is associated with begomovirus transmission, we conducted small RNA sequencing and compared the microRNA (miRNA) profiles between viruliferous whiteflies feeding on tomato plants infected with a begomovirus, tomato yellow leaf curl virus (TYLCV), and those whiteflies feeding on uninfected plants. We uncovered a comprehensive microRNA genetic regulatory system in whiteflies that may be involved in virus acquisition and transmission. Interestingly, correlating the expression profile of miRNAs and their target transcript expression in our earlier transcriptome study, we found miRNA expression was inversely correlated with predicted target gene expression in over 50% of all cases. This fundamental understanding will help identify new target sequences that could be used to improve RNA interference technology for whitefly control. These analyses could also serve as a model to study gene regulation in other systems involving arthropod transmission of viruses to plants and animals.The whitefly Bemisia tabaci (Gennadius) is a notorious insect vector that transmits hundreds of plant viruses, affecting food and fiber crops worldwide, and results in the equivalent of billions of U.S. dollars in crop loss annually. To gain a better understanding of the mechanism in virus transmission, we conducted deep sequencing of small RNAs on the whitefly B. tabaci MEAM1 (Middle East-Asia Minor 1) that fed on tomato plants infected with tomato yellow leaf curl virus (TYLCV). Overall, 160 miRNAs were identified, 66 of which were conserved and 94 were B. tabaci-specific. Among the B. tabaci-specific miRNAs, 67 were newly described in the present study. Two miRNAs, with predicted targets encoding a nuclear receptor (Bta05482) and a very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 2 (Bta10702), respectively, were differentially expressed in whiteflies that fed on TYLCV-infected versus uninfected plants. To better understand the regulatory effects of identified miRNAs and their target genes, we correlated expression profiles of miRNAs and their target transcripts and found that, interestingly, miRNA expression was inversely correlated with the expression of ~50% of the predicted target genes. These analyses could serve as a model to study gene regulation in other systems involving arthropod transmission of viruses to plants and animals.