CRT has also been described in some parasite species such as Shistosoma mansoni, Onchocerca volvulus, Necator americanus, Sorafenib Leishmania donovani and Plasmodium falciparum. However, the role that this protein might play in the parasite’s interaction with the host immediate microenvironment remains unknown. In T. cruzi, it has been suggested that CRT expressed on the parasite could play a role in enabling the pathogen to evade the host immune response by interacting with the C1q component of complement. The mechanism by which the parasite CRT interacts with host proteins to enhance the process of cellular invasion remains unknown. In this study, we hypothesize that T. cruzi uses its surface TcCRT to exploit matricellular proteins regulated by the parasite to enhance cellular infection. Pre-incubation of T. cruzi trypomastigotes with TSP-1 or NTSP significantly enhances the infection of wild type MEF compared to TSP-1 KO MEF. Blocking the TcCRT-TSP-1 interaction by pre-incubating the parasites with TcCRT antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Here we show that host TSP-1 interacts with TcCRT expressed on the surface of the parasite to enhance cellular infection. Previous studies in our laboratory showed that invasive T. cruzi trypomastigotes up-regulate the expression of TSP-1 in human coronary artery smooth muscle cells. Furthermore, knockdown of TSP-1 rendered mammalian cells less susceptible to cellular infection by T. cruzi indicating that TSP-1 plays an important role in the process of cellular infection by T. cruzi. However, the mechanism by which TSP-1 is up-regulated by the parasite modulated cellular infection is not completely known. The elucidation of molecules on the surface of T. cruzi that enhance cellular infection will advance our understanding of the molecular pathogenesis of T. cruzi infection. We anticipated that the Nterminal domain of TSP-1, which is specific to this isoform of thrombospondin, would be essential in the interaction with the parasite because it is different for all thrombospondin isoforms compared to the conserved C-terminal domain. In this study we show that TSP-1 interacts specifically with T. cruzi trypomastigote surface proteins to enhance the process of cellular infection. We used a protein-protein interaction pull down assay followed by mass spectrometric analysis to show for the first time that TSP-1 interacts with several proteins on the surface of invasive T. cruzi trypomastigotes including T. cruzi calreticulin, several T. cruzi heat shock proteins including T. cruzi HSP 60, 65 and other lower molecular weight proteins of about 30 and 36 kDa. In this study, we focused on TcCRT since it has been suggested that TSP-1 interacts with human calreticulin to signal mammalian cells. Therefore our observations agree with previous findings that TSP-1 interacts with host CRT.