Ultrasound is a non-invasive technique that has been shown to affect the cell proliferation, migration, differentiation, and promote the regeneration of cell. Our previous studies had proved that ultrasound improve natural killer (NK) cells mediated ovarian cancer therapy. To further explore the underlying mechanisms of ultrasound in NK cell and ovarian cancer cell, both cells treated by low-intensity pulsed ultrasound (LIPUS) and detected through ELISA analysis and western blot. The result demonstrated that the expression of Granzyme A, Granzyme B, perforin protein secreted by NK cells were increase. In the NK-SKOV3 co-culture system, the expression level of membrane permeability related proteins in the Wnt/β-catenin pathway of SKOV3 cells increased. This study presented cellular regulation in NK cells and ovarian cancer cells after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment on NK cells.