Arthropod vectors have multiple physical and immunological barriers that impede the development and transmission of parasites to new vertebrate hosts. These barriers include the peritrophic matrix (PM), a chitinous barrier that separates the blood bolus from the midgut epithelia and modulates vector-pathogens interactions. In tsetse flies, a sleeve-like PM is continuously produced by the cardia organ located at the fore- and midgut junction. African trypanosomes, Trypanosoma brucei, must bypass the PM twice; first to colonize the midgut and secondly to reach the salivary glands (SG), to complete their transmission cycle in tsetse. However, not all flies with midgut infections develop mammalian transmissible SG infections-the reasons for which are unclear. Here, we used transcriptomics, microscopy and functional genomics analyses to understand the factors that regulate parasite migration from midgut to SG. In flies with midgut infections only, parasites fail to cross the PM as they are eliminated from the cardia by reactive oxygen intermediates (ROIs)-albeit at the expense of collateral cytotoxic damage to the cardia. In flies with midgut and SG infections, expression of genes encoding components of the PM is reduced in the cardia, and structural integrity of the PM barrier is compromised. Under these circumstances trypanosomes traverse through the newly secreted and compromised PM. The process of PM attrition that enables the parasites to re-enter into the midgut lumen is apparently mediated by components of the parasites residing in the cardia. Thus, a fine-tuned dialogue between tsetse and trypanosomes at the cardia determines the outcome of PM integrity and trypanosome transmission success.