Both T helper interleukin 17 (IL-17)-producing cells (Th17 cells) and regulatory T cells (Tregs) have been found to be increased in human tuberculous pleural effusion (TPE); however, the possible interaction between Th17 cells and Tregs in TPE remains to be elucidated. The objective of the present study was to investigate the distribution of Th17 cells in relation to Tregs, as well as the mechanism of Tregs in regulating generation and differentiation of Th17 cells in TPE. In the present study, the numbers of Th17 cells and Tregs in TPE and blood were determined by flow cytometry. The regulation and mechanism of CD39(+) Tregs on generation and differentiation of Th17 cells were explored. Our data demonstrated that the numbers of Th17 cells and CD39(+) Tregs were both increased in TPE compared with blood. Th17 cell numbers were correlated negatively with Tregs in TPE but not in blood. When naïve CD4(+) T cells were cultured with CD39(+) Tregs, Th17 cell numbers decreased as CD39(+) Treg numbers increased, and the addition of the anti-latency-associated peptide monoclonal antibody to the coculture reversed the inhibitory effect exerted by CD39(+) Tregs. This study shows that Th17/Treg imbalance exists in TPE and that pleural CD39(+) Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.