Objective Regulatory T cells (Tregs) induce immune tolerance in patients after organ transplantation. Various immunosuppressors can affect Tregs function through different mechanisms. PD-1 and TIGIT are important receptors on Tregs surface. Here, we investigated the effects of Tacrolimus and mycophenolate mofetil (MMF) on the inhibitory function of Tregs and explored the regulatory mechanism in patients after liver transplantation. Methods Thirty patients that underwent a liver transplant and 15 healthy people were enrolled. Fifteen patients received Tacrolimus only, and 15 received a combination of Tacrolimus and MMF. Tregs and effector T cells (Teffs) were isolated using magnetic beads and were mixed at different ratios of 0:1, 1:4, 1:2 and 1:1. An inhibition assay was performed by adding anti-PD-1 and anti-TIGIT when the mixture ratio was 1:1. The Tregs inhibition rate was determined and the levels of IFN-gamma and TNF-alpha were measured. Results As the ratios of Tregs to Teffs in the mixture increased, the Tregs inhibition rate increased and the levels of IFN-gamma and TNF-alpha decreased. At each mixture ratio, Tacrolimus + MMF group had the highest Tregs inhibition rate compared to Tacrolimus and control group. At the specific mixture ratio of 1:1, the addition of both anti- PD-1 and anti-TIGIT led to lower Tregs inhibition rate and higher IFN-gamma and TNF-alpha levels in all three groups as opposed to the addition of each antibody separately. Additionally, both the decrease in the Tregs inhibition rate and the increase in the IFN-gamma and TNF-alpha levels were the most for Tacrolimus + MMF group among all cases, either adding antibodies alone or mixed. Conclusion Tacrolimus and MMF enhanced the function of Tregs by synergistically affecting PD-1 and TIGIT in liver transplant patients.