Chronic hepatitis B (CHB) is characterized by functionally impaired virus-specific CD8+ T-cell responses. However, the mechanism underlying this dysfunction has not been fully clarified. We examined the role of a newly identified protein, T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3), in regulating the antiviral CD8+ T-cell response in CHB patients. Tim-3 expression on peripheral virus-specific CD8+ T cells from 20 CHB patients and 20 healthy controls was determined by flow cytometry. The phenotypes and cytokine-producing capacity were compared between Tim-3+CD8+ and Tim-3−CD8+ T cells. The impact of Tim-3 signaling on cellular proliferation and cytokine-producing capacity was also studied. Tim-3 expression on hepatitis B virus (HBV)-specific CD8+ T cells was higher than expression on cytomegalovirus (CMV)-specific CD8+ T cells. Tim-3+ CD8+ T cells exhibited proliferative senescence phenotypes and decreased cytokine production upon antigen challenge. Finally, blocking the Tim-3 pathway significantly improved proliferation and antiviral cytokine secretion of CD8+ T cells in response to HBV-specific antigen peptides. Tim-3 negatively regulates antiviral responses of CD8+ T cells isolated from CHB patients, and this response is reversed by blocking the Tim-3 pathway.