Natural regulatory T cells (nTregs) are known to increase during chronic infection or at the site tumor, though the exact mechanisms that contribute to their accumulation and mode of action remain unclear. We used flow cytometry and microarray analyses to delineate the phenotype of nTregs and their role in modulating T cell and antigen presenting cell (APC) function in the setting of chronic infection. Using cells from 18 filaria-infected (Fil+) and 19 filaria-uninfected (Fil-) subjects, we found that the frequencies of nTreg expressing CTLA-4, GITR, LAG-3, and IL-10 were significantly higher in Fil+ compared with Fil- subjects. Microarray analysis revealed that, compared with those from Fil-, nTreg populations in Fil+ subjects were more heterogeneous and had higher expression of IL-10, CCL-4, IL-29 and CTLA-4, molecules that have been implicated in immune suppression. Moreover, the nTregs from Fil+ subjects had markedly upregulated activation-induced apoptotic genes with concomitant down regulation of cell survival genes. However, these activated nTregs from Fil+ subjects did not significantly affect cytokine production by APCs. To determine if nTregs directly modulate APC function, we modeled an in vitro culture system with cells from normal individuals. In this system, in response to antigen or anti-CD3, nTregs did inhibit APC production of IL-12-, CXCL-9, and CXCL-10, but did so indirectly through effector T cells. Taken together, our results suggest that in filarial infection, the expanded nTreg populations are heterogeneous, short-lived, activated and express regulatory molecules that modulate cytokine production by APC indirectly through an effector T cell-dependent mechanism.