Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-alpha (hCK alpha) plays in HCV replication remains elusive. Here, we first showed that hCK alpha activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy alpha and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCK alpha colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER)alpha nd that HCV infection increased hCK alpha localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCK alpha on the ER, and hCK alpha activity was required for effective trafficking of hCK alpha and NS5A to the ER. Coimmunoprecipitation showed that hCK alpha was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCK alpha silencing or treatment with CK37, an hCK alpha activity inhibitor, abolished HCV-inducedMWformation. In addition, hCK alpha depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCK alpha activity was not essential for the binding of NS5A to hCK alpha or NS5B. These findings demonstrate that hCK alpha forms a complex with NS5A and that hCK alpha activity enhances the targeting of the complex to the ER, where hCK alpha protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-alpha (hCK alpha) acts as an essential host factor for HCV RNA replication. A fraction of hCK alpha colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCK alpha, and hCK alpha activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCK alpha abrogates MW formation. Moreover, hCK alpha is recruited by NS5A independent of hCK alpha activity, presumably through binding to NS5A D1. hCK alpha activity then mediates the ER targeting of the hCK alpha-NS5A complex. On the ER membrane, hCK alpha protein, per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCK alpha-targeted anti-HCV therapeutics.