Globally suppressed T-cell function has been described in many patients with cancer to be a major hurdle for the development of clinically efficient cancer immunotherapy. Inhibition of antitumor immune responses has been mainly linked to inhibitory factors present in cancer patients. More recently, increased frequencies of CD4⁺CD25^hi regulatory T cells (T_reg cells) have been described as an additional mechanism reducing immunity. We assessed 73 patients with B-cell chronic lymphocytic leukemia (CLL) and 42 healthy controls and demonstrated significantly increased frequencies of cytotoxic T lymphocyte-associated protein 4 (CTLA4⁺)–, Forkhead box P3 (FOXP3⁺)–, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR⁺)–, CD62L⁺–, transforming growth factor β1 (TGF-β1⁺)–, interleukin 10 (IL-10⁺)–T_reg cells in patients with CLL, with highest frequencies in untreated or progressing patients presenting with extended disease. Most surprisingly, in the majority of patients with CLL treated with fludarabine-containing therapy regimens the inhibitory function of T_reg cells was decreased or even abrogated. In addition, frequencies of T_reg cells were significantly decreased after therapy with fludarabine. In light of similar findings for cyclophosphamide the combination of fludarabine and cyclophosphamide might be further exploited in strategies reducing immunosuppression prior to cancer immunotherapy.