Pentamidine reduces viability of Trypanosoma cruzi and decreases the parasite infection in vitro and in vivo. Also, pentamidine inhibits the polyamine transport in the parasite. •We studied the effect of pentamidine against Trypanosoma cruzi both in vitro and in vivo.•Pentamidine decreases the viability of isolated trypomastigotes of T. cruzi.•Pentamidine decreases the parasite burden in T. cruzi-infected cells.•Pentamidine improves the disease outcome in T. cruzi-infected mice.•Pentamidine inhibits the polyamine transport in isolated parasites. Pentamidine is an antiprotozoal and fungicide drug used in the treatment of leishmaniasis and African trypanosomiasis. Despite its extensive use as antiparasitic drug, little evidence exists about the effect of pentamidine in Trypanosoma cruzi, the etiological agent of Chagas’ disease. Recent studies have shown that pentamidine blocks a polyamine transporter present in Leishmania major; consequently, its might also block these transporters in T. cruzi. Considering that T. cruzi lacks the ability to synthesize putrescine de novo, the inhibition of polyamine transport can bring a new therapeutic target against the parasite. In this work, we show that pentamidine decreases, not only the viability of T. cruzi trypomastigotes, but also the parasite burden of infected cells. In T. cruzi-infected mice pentamidine decreases the inflammation and parasite burden in hearts from infected mice. The treatment also decreases parasitemia, resulting in an increased survival rate. In addition, pentamidine strongly inhibits the putrescine and spermidine transport in T. cruzi epimastigotes and amastigotes. Thus, this study points to reevaluate the utility of pentamidine and introduce evidence of a potential new action mechanism. In the quest of new therapeutic strategies against Chagas disease, the extensive use of pentamidine in human has led to a well-known clinical profile, which could be an advantage over newly synthesized molecules that require more comprehensive trials prior to their clinical use.