Abstract We studied the involvement of the p38 signaling pathway in the interferon (IFN)-α-mediated antiviral activity toward hepatitis C virus (HCV) using HCV subgenomic replicon cells. When the cells were treated with IFN-α in the presence of p38 inhibitor, the suppressive effect of IFN-α on replicon RNA was reduced. Inhibition of p38 had almost no influence on phosphorylation of signal transducer and activator transcription factor 1 (STAT1) and interferon stimulatory response element-dependent gene expression after IFN-α treatment. This indicates that the anti-HCV activity through p38 may be independent of the Janus kinase-STAT pathway. Treatment with the inhibitor of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) showed the same level of reduction in the IFN-α-mediated anti-HCV activity as that with the p38 inhibitor. Thus, MK2 may also be responsible for the anti-HCV activity through p38. In conclusion, the p38-MK2 signaling pathway may be substantially involved in the IFN-α-mediated anti-HCV activity.