<i>Objective:</i> The aim of this study was to evaluate whether gene expression of Toll-like receptor (TLR)-3, TLR4, TLR7 and TLR9 was impaired in patients with chronic HIV-1 infection who were failing to respond to antiretroviral therapy. <i>Methods:</i> Transcripts encoding TLRs were assayed by quantitative real time RT-PCR on peripheral blood mononuclear cells derived from patients with HIV-1 infection who were responding or not responding to antiretroviral therapy and healthy control subjects. <i>Results:</i> Chronic HIV-1-infected patients who failed to respond to therapy showed reduced expression of TLRs 3, 4 and 9, together with increased expression of TLR7, as compared to healthy subjects. Moreover, a trend towards a higher expression of TLR3 and TLR9 was observed in responder patients compared with non-responders. In addition, we found lower levels of TRLs 3, 7 and 9 in patients with high levels of HIV-1 RNA compared to those with lower levels of viremia. <i>Conclusions:</i> These findings demonstrate that in chronic HIV-1-positive patients who were failing to respond to the therapy, there were substantial changes in TLRs expression. This is likely to be an important determinant of the clinical course of HIV-1 infection.