Rhodnius prolixus is one of the most important vectors of Chagas’ disease caused by the parasite Trypanosoma cruzi. Vector control is the principal combat method of this disease. It is inside of intestine that T. cruzi completes his life cycle to become infectious. On this work a 2D protein map of R. prolixus intestine was performed. On the 2D-map, soluble proteins from crop were separated by two-dimensional gel electrophoresis using 18cm IPG strips pH 4-7 and 12.5% SDS-PAGE gels. After Coomassie blue staining all visible spots were excised, proteins were trypsin digested and analyzed by nano-LC MS/MS. Peptides were identified using in-house Mascot software (Matrixscience) and searched against genomic and transcriptomic databases. 692 spots were picked. Mascot analysis resulted in a total of 476 spots (69%) with a set of 490 proteins identified. These data provide experimental evidence for R. prolixus proteins that were predicted by genome and transcriptome. These proteomic data, together with transcriptomic and genomic information, will contribute in the comprehension of the physiology of R. prolixus midgut.