Background Farm exposure protects against development of allergies early in life. At 4.5 years, protection against asthma by farm-milk exposure was partially mediated by regulatory T cells (Tregs). The aim of this study was to investigate the critical time window of the 'asthma-protective' farm effect via Tregs during childhood immune maturation. Methods Tregs were assessed longitudinally at 4.5 and 6 years in 111 children (56 farm and 55 reference children) from the PASTURE/ EFRAIM birth cohort (flow cytometry). Peripheral blood mononuclear cells were cultured unstimulated (U), with phorbol 12-myristate 13-acetate/ionomycin ( PI) or lipopolysaccharide ( LPS), and stained for Tregs ( CD4+ CD25high FOXP3upper20%). mRNA expression of Treg/Th1/Th2/Th17-associated cell markers was measured ex vivo. Suppressive capacity of Tregs on effector cells and cytokines was assessed. Detailed questionnaires assessing farm exposures and clinical phenotypes from birth until age 6 years were answered by the parents. Results Treg percentage before and after stimulation and FOXP3 mRNA expression ex vivo decreased from age 4.5 to 6 years ( P(U, LPS) < 0.001; P( PI) = 0.051; P( FOXP3) < 0.001). High vs low farm-milk and animal-stable exposure was associated with decreased LPS-stimulated Treg percentage at age 6 years ( P( LPS) = 0.045). Elevated LPS-stimulated-Treg percentage at age 6 was associated with increased risk of asthma ( aOR = 11.29, CI: 0.96-132.28, P = 0.053). Tregs from asthmatics vs nonasthmatics suppressed IFN-γ ( P = 0.015) and IL-9 ( P = 0.023) less efficiently. mRNA expression of Th1/Th2/Th17-associated cell markers decreased between 4.5 and 6 years ( P < 0.001). Conclusions Tregs at the age of 6 years were decreased with farm exposure and increased within asthmatics, opposite to age 4.5 years. This immunological switch defines a critical 'time window' for Treg-mediated asthma protection via environmental exposure before age 6 years. [ABSTRACT FROM AUTHOR]