Chronic hepatitis C virus (HCV) infection is characterized by high interindividual variability in response to pegylated interferon and ribavirin. A genetic polymorphism on chromosome 19 (rs12979860) upstream of interferon-3 (IFN3) is associated with a twofold change in sustained virologic response rate after 48weeks of treatment with pegylated interferon/ribavirin in HCV genotype 1 (GT1) treatment-naive patients. We conducted epigenetic analysis on the IFN3 promoter to investigate whether DNA methylation is associated with response to HCV therapy. DNA samples from HCV GT1-infected subjects receiving an interferon-free paritaprevir-containing combination regimen (N=540) and from HCV-uninfected, healthy controls (N=124) were analysed for IFN3 methylation levels. Methylation was strongly associated with rs12979860 allele status whether adjusting for HCV status (r=65.0%, 95% CI: [60.2%, 69.5%]), or not (r=64.4%), both with P<2.2x10(-16). In HCV GT1-infected subjects, C/C genotypes had significantly lower methylation levels relative to C/T or T/T genotypes (P<1x10(-14)), with each T allele resulting in a nine-unit increase in mean methylation level. Methylation levels did not correlate with response in subjects treated for 12 or 24 weeks. However, non-C/C subjects with higher methylation levels were more likely to relapse when treatment duration was 8weeks. This analysis suggests that methylation status of the IFN3 promoter region may be a useful parameter that identifies patients more likely to relapse following HCV therapy; however, continuing therapy for a sufficient duration can overcome this difference. These findings may provide mechanistic insight into the role of IFN3 genetic variants in HCV treatment response.