CD4+CD25+ regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses in order to inhibit pathogenic damage resulting from over activation of the immune system. In human immunodeficiency virus-1 (HIV-1) infection, suppression of the immune response by Tregs appears to play an opposing role that promotes chronic viral infection. Treg expansion is known as a marker of the severity of HIV infection and as a potential prognostic marker of disease progression. HIV-1 Nef is one of the earliest expressed viral regulatory genes whose expression may play an important role in regulating Treg cells. We established a THP-1 cell line stably expressing HIV-1 Nef and showed that Nef protein was a potent factor for increasing Treg numbers in vitro. We further found that TLR2 plays a critical role in the increase in Treg cells induced by Nef using TLR2-specific siRNA. Our results suggest new strategies for therapeutic and preventive interventions of HIV infection. J. Cell. Biochem. 112: 3515–3524, 2011. © 2011 Wiley Periodicals, Inc.