Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is involved in many essential biological processes in T. cruzi, its transport and metabolism are interesting drug targets. Four synthetic proline analogues (ITP-1B/1C/1D/1G) were evaluated as inhibitors of proline transport mediated through the T. cruzi proline permease TcAAAP069. The trypanocidal activity of the compounds was also assessed. The compounds ITP-1B and ITP-1G inhibited proline transport mediated through TcAAAP069 permease in a dose-dependent manner. The analogues ITP-1B, -1D and -1G had trypanocidal effect on T. cruzi epimastigotes with IC50 values between 30 and 40μM. However, only ITP-1G trypanocidal activity was related with its inhibitory effect on TcAAAP069 proline transporter. Furthermore, this analogue strongly inhibited the parasite stage differentiation from epimastigote to metacyclic trypomastigote. Finally, compounds ITP-1B and ITP-1G were also able to inhibit the transport mediated by other permeases from the same amino acid permeases family, TcAAAP. It is possible to design synthetic amino acid analogues with trypanocidal activity. The compound ITP-1G is an interesting starting point for new trypanocidal drug design which is also an inhibitor of transport of amino acids and polyamines mediated by permeases from the TcAAAP family, such as proline transporter TcAAAP069 among others. The Trypanosoma cruzi amino acid transporter family TcAAAP constitutes a multiple and promising therapeutic target for the development of new treatments against Chagas disease. •Synthetic proline analogues are proposed as new trypanocidal drugs.•One of this analogues inhibits amino acid transport and has trypanocidal action.•The TcAAAP transporter family constitutes a promising multiple therapeutic target.