The current study was aimed at developing a one‐way mixed leucocyte culture–enzyme‐linked immunospot (MLC‐ELISPOT) assay for the study of CD4⁺ CD25⁺ regulatory T (T_reg) cells and applying this method in the study of antifetal immune reactions during human pregnancy. Twenty‐one pregnant women and the corresponding fathers‐to‐be, and 10 non‐pregnant control women and men, participated in the study. CD4⁺ CD25⁺ cells were isolated from peripheral blood mononuclear cells (PBMC) by immunomagnetic selection. Maternal/control PBMC were stimulated with paternal or unrelated PBMC in MLC. Secretion of interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) from responder cells, with or without the presence of autologous T_reg cells, was analysed by ELISPOT. PBMC from pregnant women showed increased secretion of IL‐4 compared to controls. In pregnant and non‐pregnant controls, T_reg cells suppressed IFN‐γ reactivity against paternal and unrelated alloantigens. Interestingly, T_reg cells suppressed IL‐4 secretion against paternal but not unrelated alloantigens during pregnancy. We have successfully developed a model for studying T_reg cells in antifetal cytokine reactions during pregnancy. Results indicate that T_reg cells contribute to strict regulation of both T helper type 1‐like and type 2‐like antifetal immune reactions. Interestingly, T helper type 2‐like cells specific to unrelated alloantigens are able to escape the suppression of T_reg cells, which would allow for IL‐4, alongside CD4⁺ CD25⁺ T_reg cells, to control potentially detrimental IFN‐γ reactions during pregnancy.