CD4+CD25+ regulatory T cells are involved in the prevention of autoimmune diseases and in tumor-induced tolerance. We previously demonstrated in tumor-bearing rodents that one injection of cyclophosphamide could significantly decrease both numbers and suppressive functions of regulatory T cells, facilitating vaccine-induced tumor rejection. In humans, iterative low dosing of cyclophosphamide, referred to as "metronomic" therapy, has recently been used in patients with advanced chemotherapy resistant cancers with the aim of reducing tumor angiogenesis. Here we show that oral administration of metronomic cyclophosphamide in advanced cancer patients induces a profound and selective reduction of circulating regulatory T cells, associated with a suppression of their inhibitory functions on conventional T cells and NK cells leading to a restoration of peripheral T cell proliferation and innate killing activities. Therefore, metronomic regimen of cyclophosphamide does not only affect tumor angiogenesis but also strongly curtails immunosuppressive regulatory T cells, favoring a better control of tumor progression. Altogether these data support cyclophosphamide regimen as a valuable treatment for reducing tumor-induced immune tolerance before setting to work anticancer immunotherapy.