The 3' untranslated region (3'UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1 alpha. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle.