MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, are key molecules in many biological and metabolic processes of plant growth, development and stress response via targeting mRNAs. The phloem-feeding insect whitefly Bemisia tabaci (Hemiptera, Aleyrodidae) is a serious pest that causes devastating harm to agricultural production worldwide. However, the function of host miRNAs in the response to whitefly infestation remains unclear. Here, we sequenced the small RNA and degradome of tobacco (Nicotiana tabacum L.), after and before infestation by B. tabaci. We identified 1291 miRNAs belonging to 138 miRNA families including 706 known miRNAs and 585 novel miRNAs. A total of 47 miRNAs were differentially expressed, of which 30 were upregulated and 17 were downregulated by whitefly exposure. Then, computational analysis showed that the target genes of differential miRNAs were involved in R gene regulation, plant innate immunity, plant pathogen defense, the plant hormone signal pathway and abiotic stress tolerance. Furthermore, degradome analysis demonstrated that 253 mRNAs were cleaved by 66 miRNAs. Among them, the targets cleaved by upregulated miR6025, miR160, miR171, miR166 and miR168 are consistent with our prediction, suggesting that pathogen-related miRNAs may function in plant defense against whitefly. Moreover, our results show that plant miRNA response and miRNA-mediated post-transcriptional regulation for phloem-feeding insect infestation are similar to pathogen invasion. Our study provides additional data to further elucidate how host plants respond and defend the phloem-feeding insects.