In trypanosomes regulation of gene expression occurs mainly at the post-transcriptional level. Pumilio proteins are RNA-binding proteins that modulate gene expression in lower and higher eukaryotes. Here we present the characterization of TcPUF6, a member of the Pumilio family in Trypanosoma cruzi. TcPUF6 is expressed in the different life cycle forms of the parasite showing no clear stage specific regulation and it is localized to multiple discrete foci in the cytoplasm of epimastigotes. The recombinant TcPUF6 fusion protein specifically binds to the Drosophila hunchback NRE (nanos response element). TcPUF6 conserves functional properties that characterize the Pumilio family throughout evolution.