JTK-853 is a novel piperazine derivative nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase. JTK-853 showed potent inhibitory activity against genotype 1 HCV polymerase, with a 50% inhibitory concentration in the nanomolar range, and showed potent antiviral activity against the genotype 1b replicon, with a 50% effective concentration of 0.035 μM. The presence of human serum at up to 40% had little effect on the antiviral activity of JTK-853. Structure analysis of HCV polymerase with JTK-853 revealed that JTK-853 associates with the palm site and β-hairpin region of HCV polymerase, and JTK-853 showed decreased antiviral activity against HCV replicons bearing the resistance mutations C316Y, M414T, Y452H, and L466V in the palm site region of HCV polymerase. JTK-853 showed an additive combination effect with other DAAs (direct antiviral agents), such as nucleoside polymerase inhibitor, thumb pocket-binding nonnucleoside polymerase inhibitor, NS5A inhibitor, and protease inhibitor. Collectively, these data demonstrate that JTK-853 is a potent and novel nonnucleoside palm site-binding HCV polymerase inhibitor, suggesting JTK-853 as a potentially useful agent in combination with other DAAs for treatment of HCV infections.