Diatraea saccharalis constitutes a threat to the sugarcane productivity, and obtaining borer tolerant cultivars is an alternative method of control. Although there are studies about the relationship between the interaction of D. saccharalis with sugarcane, little is known about the molecular and genomic basis of defense mechanisms that confer tolerance to sugarcane cultivars. Here, we analyzed the transcriptional profile of two sugarcane cultivars in response to borer attack, RB867515 and SP80-3280, which are considered tolerant and sensitive to the borer attack, respectively. A sugarcane genome and transcriptome were used for read mapping. Differentially expressed transcripts and genes were identified and termed to as DETs and DEGs, according to the sugarcane database adopted. A total of 745 DETs and 416 DEGs were identified (log₂|ratio| > 0.81; FDR corrected P value ≤ 0.01) after borer infestation. Following annotation of up- and down-regulated DETs and DEGs by similarity searches, the sugarcane cultivars demonstrated an up-regulation of jasmonic acid (JA), ethylene (ET), and defense protein genes, as well as a down-regulation of pathways involved in photosynthesis and energy metabolism. The expression analysis also highlighted that RB867515 cultivar is possibly more transcriptionally activated after 12 h from infestation than SP80-3280, which could imply in quicker responses by probably triggering more defense-related genes and mediating metabolic pathways to cope with borer attack.