The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B-ESR1C and ESR1C-ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B-ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.