Crystal structures of a genogroup II.4 human norovirus (NV) polymerase bound to an RNA primer-template duplex and the substrate-analogue 2'-amino-2'-deoxycytidine-5'-triphosphate (ACT) have been determined to 1.8 A resolution. The alteration of the substrate-binding site that is required to accommodate the 2'-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for ACT suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.