Human immunodeficiency virus/hepatitis C virus (HIV/HCV) co-infection is still a worldwide problem. CD8+ T lymphocytes are essential in the immune system to suppress viral and infected cells. The aim of this study was to compare HIV-specific CD8+T lymphocyte responses in HIV/HCV co-infected and HIV mono-infected patients. Three groups of 23 patients were recruited from Chulalongkorn Hospital. Eleven patients were HIV mono-infected, eight were HIV mono-infected (they received GPO-VIR), and four patients were HIV/HCV co-infected. IFN gamma secreting CD8+T lymphocytes to the HIV-1 Gag and Env peptides were measured using an ELISpot assay. To determine the levels of Granzyme A, Granzyme B and perforin, all samples were measured using flow cytometry. The results showed that there was a significant decrease in the level of HIV-1 Env-specific CD8+ T lymphocytes in the HIV/HCV co-infected patients. This study found that patients with HIV/HCV co-infection have a slightly lower specific CD8+ T lymphocytes response compared with other HIV and HCV patients.