CD4+CD25high regulatory T cells (Tregs) control cellular immune responses and maintain peripheral tolerance. We investigated whether TLR2 ligands are able to abrogate Treg-induced suppression in humans based on different reports about effects of triacylated lipopeptide Pam3CSK4 in mice. Pretreatment of human Tregs with a mixture of TLR2 ligands Pam2CSK4, FSL-1, and Pam3CSK4 reduced the Treg-mediated suppression of CD4+CD25− responder T cells in the majority of the analyzed donors. Differential effects of individual TLR2 ligands are explained by usage of different TLR2 heterodimers in the recognition of Pam2CSK4, FSL-1, and Pam3CSK4. In contrast to the murine system, TLR2 ligand-mediated abrogation of human Treg function was not associated with a downregulation of FoxP3 transcription factor. Furthermore, our results excluded an effect of TLR2 ligands on granzyme A/B release by human Tregs as a potential mechanism to abolish Treg-mediated suppression. Our data suggest that a downregulation of p27Kip1 and restoration of Akt phosphorylation in human Tregs pretreated with TLR2 ligands result in a reversal of suppression on responder T cells. Moreover, our data indicate that a mixture of TLR2 ligands can be used to modulate human Treg activity.