Plant viruses in agricultural crops are of great concern worldwide, and over 75% of them are transmitted from infected to healthy plants by insect vectors. Tomato yellow leaf curl virus (TYLCV) is a begomovirus, which is the largest and most economically important group of plant viruses, transmitted by the whitefly Bemisia tabaci. The circulation of TYLCV in the insect involves complex insect-virus interactions, whereas the molecular mechanisms of these interactions remain ambiguous. The insect gut as a barrier for viral entry and dissemination is thought to regulate the vector specificity. However, due to its tiny size, information for the responses of whitefly gut to virus infection is limited. We investigated the transcriptional response of the gut of B. tabaci Middle East-Asia Minor 1 species to TYLCV infection using Illumina sequencing. A total of 5207 differentially expressed genes (DEGs) between viruliferous and non-viruliferous whitefly guts were identified. Enrichment analyses showed that cargo receptor and ATP-binding cassette (ABC) transporters were enriched in DEGs, and might help the virus to cross gut barrier. TYLCV could perturb cell cycle and DNA repair as a possible result of its replication in the whitefly. Our data also demonstrated that TYLCV can activate whitefly defense responses, such as antimicrobial peptides. Meanwhile, a number of genes involved in intracellular signaling were activated by TYLCV infection. Our results reveal the complex insect-virus relationship in whitefly gut and provide substantial molecular information for the role of insect midguts in virus transmission.