Interactions between Trypanosoma cruzi and the extracellular matrix play an important role in cellular invasion. Here we show that T. cruzi increases the levels of thrombospondin-1 (TSP-1) expression in host cells during early infection. Stable RNA interference of host cell TSP-1 knocks down the levels of TSP-1 transcripts and protein expression in mammalian cells causing inhibition of T. cruzi infection. Addition of TSP-1 to these cells restores infection. Thus, host TSP-1, regulated by the parasite, plays a crucial role in early infection. This is the first report showing that a human parasite modulates TSP-1 expression to facilitate infection.