Background & Aims Spontaneous resolution of hepatitis C virus (HCV) infection depends upon a broad T cell response to multiple viral epitopes. However, most patients fail to clear infections spontaneously and develop chronic disease. The elevated number and function of CD3+CD4+CD25+FoxP3+ regulatory T cells (T(reg)) in HCV-infected patients suggest a role of Treg cells in impaired viral clearance. The factors contributing to increased Treg cell activity in chronic hepatitis C cases remain to be delineated. Methods Immunoinformatics tools were used to predict promiscuous, highly-conserved HLA-DRB1-restricted immunogenic consensus sequences (ICS), each composed of multiple T cell epitopes. These sequences were synthesized and added to cultures of peripheral blood mononuclear cells (PBMCs), derived from patients who resolved HCV infection spontaneously, patients with persistent infection, and non-infected individuals. The cells were collected and following 5 days incubation, quantified and characterized by flow cytometry. Results One immunogenic consensus sequence (ICS), HCV-G1-p7-794, induced a marked increase in Treg cells in PBMC cultures derived from infected patients, but not in patients who spontaneously cleared HCV or in non-infected individuals. An analogous human peptide (p7-794), on the other hand, induced a significant increase in Treg cells among PBMCs derived from both HCV-infected and non-infected individuals. JanusMatrix analyses determined that HCV-G1-p7-794 is comprised of Treg cell epitopes that exhibit extensive cross-reactivity with the human proteome. Conclusions A virus-encoded peptide (HCV-G1-p7-794) with extensive human homology activates cross-reactive CD3+CD4+CD25+FoxP3+ natural Treg cells, which potentially contributes to immunosuppression and to the development of chronic hepatitis C. © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.