Polymorphisms within and upstream of Interferon-lambda (IFN-λ) gene 3 (IFN-λ3, IL-28B gene) are strongly associated with the clearance of Hepatitis C virus (HCV) in chronically infected patients treated with interferon-alpha (IFN-α). Human blood and liver dendritic cells (DC) expressing CD141 and Clec9A are known to produce IFN-λ in response to dsRNA and in particular, to HCV. In this study we enumerated the CD141⁺ DC in the peripheral blood of a young cohort of injecting drug users (IDUs), with chronic HCV infection, not undergoing treatment, and assayed their ability to produce IFN-λ. DCs from chronic HCV-infected IDUs expressing the protective IL-28B genotype (rs8099917, TT and rs12979860, CC) were compared to those expressing IL-28B rs8099917, GT or GG and rs12979860 CT or TT. Regardless of the IFN-λ genotype, IFN-λ production was reduced from the total PBMCs of chronically infected HCV donors. This reduction was commensurate with a common loss of CD141⁺ DC from the blood of all chronically infected individuals. However, the production of IFN-λ was increased at least 2-fold by the pre-treatment of PBMC with IFN-α in healthy subjects and in chronically infected patients, regardless of genotype. Thus those cells most efficient at IFN-λ production, CD141⁺ DC, although substantially reduced in blood in the cohort we have examined, respond to IFN-α with increased IFN-λ production, regardless of the IL-28B genotype.