Chronic infection by Hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remain largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3' nontranslated region (3'NTR) of the (+) strand HCV genome are essential for initiation of (-) strand synthesis. The RNA signals within the highly conserved 3' NTR may be the site for recruiting cellular factors which mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our lab used in conjunction with LC/MS/MS mass spectrometry has allowed us to positively identify more than 70 cellular proteins that interact with the 3'NTR (+) of HCV. Binding of these cellular proteins was not competed out by a 10-fold excess of nonspecific competitor RNA. With few exceptions, all of the identified cellular proteins are RNA binding proteins whose reported cellular functions provide unique insights into host cell-virus interactions and possible mechanisms influencing HCV replication and HCV-associated pathogenesis. SiRNA mediated silencing of selected 3' NTR-binding proteins in an HCV replicon cell line reduced replicon RNA to undetectable levels, suggesting important roles for these cellular factors in HCV replication.