Powdery mildew, caused by Sphaerotheca fuliginea (Sf), is a widely distributed and destructive disease of greenhouse and field-grown cucumber plants and causes great yield loss. The objective of this research is to tentatively identify proteins that are differentially expressed in cucumber and are involved in modulating resistance to Sf-inoculation. We comparatively analyzed proteins differentially expressed in Sf-inoculated cucumber leaves using a pair of sister lines, B21-a-2-2-2 (highly susceptible) and B21-a-2-1-2 (highly resistant). To eliminate the interference of ribulose-1,5-bisphosphate carboxylase with low-abundance proteins, total proteins were pre-fractionated by 24 % polyethylene glycol (PEG) and the proteins from supernatant were analyzed by 2-DE. We were successful in establishing the identities of 20 proteins and those identified from the resistant line included proteins involved in metabolic, regulatory, and defense pathways. Our findings are discussed within the context of C. sativus–S. fuliginea interaction and tolerance to this pathogen. The results suggest that the resistance in cucumber is closely related to the enhancement of its primary metabolism, and ethylene modulated signaling in cucumber defense responses against powdery mildew, then defense-related proteins can be up-regulated as a result of altered gene expression.