Immunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC). However, CD4+CD25+Foxp+ regulatory T cells, which suppress potential antigen-specific T-cell responses, are increased in patients with HCC and might impair the effect of an immune-based therapeutic approach. In this study, we demonstrate that depletion of regulatory T cells in vitro unmasks α-fetoprotein-specific T-cell responses in HCC patients. On the basis of these results, we performed a clinical trial, in which 13 patients with advanced HCC ineligible for any other type of treatment were treated with 150, 250, or 350 mg/m2 cyclophosphamide on day 1 and 29 to suppress regulatory T cells in these patients (NCT00396682). The primary end point of this trial was regulatory T-cell number and function. Low-dose cyclophosphamide treatment (150 and 250 mg/m2) induced a decrease in the absolute and relative frequency of CD4+CD25+Foxp+ regulatory T cells in peripheral blood on days 8 and 29. Suppressor function of regulatory T cells was impaired after treatment of patients with 250 mg/m2 on days 8 and 21. Finally, α-fetoprotein-specific T-cell responses were unmasked in 6/13 treated patients. In summary, systemic treatment of HCC patients with low-dose cyclophosphamide decreases the frequency and suppressor function of circulating CD4+CD25+Foxp+ regulatory T cells in peripheral blood and could be used in combination with immunotherapeutic approaches in HCC.