The causal agent of red stripe, Acidovorax avenae subsp. avenae (Aaa), can incite significant economic losses during sugarcane production. Proteomics is a useful tool for the comprehensive identification of defense response proteins in plants. In this study, isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics was used to identify proteins that were produced in sugarcane in defense to Aaa in two Saccharum spp. cultivars: ROC22 (resistant to red stripe) and MT11-610 (susceptible). Defense response was evaluated at 0 h (mock-inoculation) and 24 h after inoculation with the bacterial pathogen, and 10,502 high-confidence proteins and 1962 differentially expressed proteins (DEPs) were identified. These DEPs included 1027 proteins (671 upregulated, 354 downregulated) in ROC22 and 1130 proteins (566 upregulated, 564 downregulated) in MT11-610. Gene ontology analysis revealed that most DEPs were clustered in the metabolic, single-organism metabolic, and oxidation-reduction processes, and in catalytic activity. In a KEGG analysis, these proteins belonged to common pathways such as secondary metabolite biosynthesis and biosynthesis of phenylpropanoids. Thirty-six DEPs were identified, especially epoxide hydrolase 1, dynamin-related protein 1C-like, cytochrome oxidase subunit I, FK506-binding protein, acetoacetyl CoA thiolase isoform 2, anthocyanidin 3-o-glucosyltransferase, ubiquitin carboxyl-terminal hydrolase 4, protein disulfide isomerase-like protein, and jumonji-like transcription factor family protein. These nine proteins were characterized by enhanced expression with =1.5-fold increases in resistant ROC22 versus susceptible MT11-610, and are therefore biomarker candidates in the sugarcane response against Aaa. These data could be very useful in molecular breeding for sugarcane resistance to red stripe.