Trypanosoma cruzi cAMP-mediated invasion has been long described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we proved that the cAMP/Epac pathway is activated in different cells lines and, by pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP) and invasion assays using cells transfected with a constitutively active form of Rap1b (Rap1b-G12V), established the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.