The gating of charge transport through single-molecule junctions is considered a critical step towards molecular circuits but remains challenging. In this work, we report an electrostatic gating method to tune the conductance of single-molecule junctions using the scanning tunneling microscope break junction (STM-BJ) technique incorporated with a back-gated chip as a substrate. We demonstrated that the conductance varied at different applied gating voltages (Vgs). The HOMO-dominated molecules show a decrease in conductance with an increase in Vg, and the LUMO-dominated molecules show the opposite trend. The measured conductance trends with Vg are consistent with the transition voltage spectroscopy measurements. Moreover, the transmission functions simulated from density functional theory (DFT) calculations and the finite element analysis all suggest that Vg changed the energy alignment of the molecular junction. This work provides a simple method for modulating the molecular orbitals' alignment relative to the Fermi energy (Ef) of metal electrodes to explore the charge transport properties at the single-molecule scale.