Unidirectional liquid penetration plays an important role in many fields, such as microfluidic devices, biological medical, liquid printing, and oil/water separation. Although there are some progresses in the liquid unidirectional penetration using a variety of Janus membranes with anisotropic wettability, it still remains a great difficulty for single-layer Janus membranes with straight pore to balance spontaneous liquid penetration in positive direction and superior liquid resistance in the reverse direction. Herein, a liquid-assisted strategy for single-layer Janus membrane is developed, which can efficiently decrease the critical breakthrough pressure from superhydrophobic side to hydrophilic side and show little influence on that in the reverse direction. Consequently, unidirectional water penetration with high hydraulic pressure difference can be achieved. The Laplace pressure change along the thickness of the single-layer Janus membranes is further discussed, and the mechanism by which the auxiliary liquid decreases the critical breakthrough pressure is revealed. Furthermore, this Janus membrane with unidirectional water penetration “diode” performance can be used to prevent liquid backflow in intravenous transfusion. It is believed that this work can open an avenue for people to design single-layer Janus membrane with high pressure difference and find wide applications in unidirectional liquid transport.