The NS5B encoded by the hepatitis C virus genome is an approximately 25 kDa RNA-dependent RNA polymerase, which essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. NS5B has a typical ‘right hand’ polymerase structure, with catalytic sites in the base of the palm domain, surrounded by thumb and finger domains. These latter domains fully encircle the active site, creating a channel for binding to a ssRNA template. NS5B is tethered to membranes by a C-terminal peptide anchor and interacts with itself to form higher-order RdRP complexes. Together with other nonstructural viral proteins (NS3, NS4A, NS4B, and NS5A) and host factors, the NS5B RNA polymerase constitutes the active replication complexes (RC) for viral RNA replication. These proteins are directly or indirectly associated with the endoplasmic reticulum (ER)-derived structure called the “membranous web”, where replication occurs.