This study involved in-depth transcriptome analysis of the midgut of the human malaria vector, the An. gambiae mosquito, using RNA-seq by Illumina sequencing. The goal of the study was to discover new transcripts and improve the genome annotation, especially of midgut-expressed genes, as interaction of Plasmodium with this organ is critical for the malaria parasite to establish an infection. We explored the mosquito midgut transcriptome of two An. gambiae strains, the L3-5 refractory strain that was selected to melanize Plasmodium, and the An. gambiae G3 susceptible strain, both under different physiologic conditions.