Cucumber mosaic virus (CMV) is among the most important plant virus infections, inducing a variety of disease symptoms. However, the molecular mechanisms underlying plant responses to CMV infection remain poorly understood. In this study, we performed RNA sequencing analysis of tolerant (Taiyan8) and susceptible (NC82) tobacco cultivars on CMV-infected plants, using mock-inoculated plants as a control. The propagation of CMV in inoculated leaves did not show obvious difference between two cultivars, whereas virus accumulation in systemic leaves of Taiyan8 was smaller than those of NC82 at the same time point. We observed 765 and 1,011 differentially expressed genes (DEGs) in Taiyan8 and NC82, respectively, in CMV-inoculated leaves. DEGs related to reactive oxygen species, salicylic acid signal transduction, and plant-pathogen interaction were upregulated or downregulated in Taiyan8, which indicates that defense response pathways to CMV were activated in the tolerant cultivar. In addition, we identified several DEGs related to disease defense and stress resistance showing opposing expression patterns in the two cultivars. Our comparative transcriptome analysis will improve our understanding of the mechanisms of CMV tolerance in plants, and will be of great importance in the molecular breeding of CMV- tolerant genotypes.