The pregnancy hormone human chorionic gonadotropin (hCG) reportedly modulates innate and adaptive immune responses and contributes thereby to fetal survival. More precisely, hCG has been shown to support human regulatory T cell (Treg cell) homing into the fetal-maternal interface and enhance the number and function of Treg cells in murine pregnancy. Here, we aimed to study whether hCG and hCG-producing human trophoblast cell lines induce Treg cells from CD4 FOXP3⁻ T cells and promote T cell suppressive activity. CD4 FOXP3⁻ T cells were isolated from peripheral blood of normal pregnant women and cultured in the presence of hCG-producing (JEG-3, HTR-8) and nonproducing (SWAN-71) cell lines. To confirm the participation of hCG in Treg cell conversion, the experiments were performed in the presence of anti-hCG and additional experiments were run with recombinant or urine-purified hCG. After culture, the number of CD4 FOXP3 Treg cells as well as the suppressive capacity of total T cells was assessed. The hCG-producing JEG-3 cells as well as recombinant and urine-purified hCG induced CD4 FOXP3 Treg cells from CD4 FOXP3⁻ T cells. Blockage of hCG impaired Treg cell induction. Moreover, hCG-producing JEG-3 cells increased suppressive activity of CD4 FOXP3⁻ T cells through an antigen-independent pathway. Our results propose another mechanism through which hCG modulates the female immune system during pregnancy in favor of the fetus.