We previously demonstrated that morphine enhances hepatitis C virus (HCV) replication in human hepatic cells. Here we describe the impact of morphine withdrawal (MW), a recurrent event during the course of opioid abuse, on HCV replicon expression in human hepatic cells. MW enhanced both viral RNA and protein expression in HCV replicon cells. Blocking opioid receptors by treatment with naloxone after morphine cessation (precipitated withdrawal, PW) induced greater HCV replicon expression than MW. Investigation of the mechanism responsible for MW- or PW-mediated HCV enhancement showed that both MW and PW inhibited the expression of endogenous interferon-alpha (IFN-alpha) in the hepatic cells. This down-regulation of intracellular IFN-alpha expression was due to the negative impact of MW or PW on IFN-alpha promoter activation and on the expression of IFN regulatory factor 7 (IRF-7), a strong transactivator of the IFN-alpha promoter. In addition, both MW and PW inhibited the anti-HCV ability of recombinant IFN-alpha in the hepatic cells. These in vitro observations support the concept that opioid abuse favors HCV persistence in hepatic cells by suppressing IFN-alpha-mediated intracellular innate immunity and contributes to the development of chronic HCV infection.