Abstract: The microstreaming induced by low-intensity pulsed ultrasound (LIPUS) causes the shear stress, which has been often used to enhance the permeability of cells' membrane. In the present study, to simulate the in vivo microenvironment, three-dimensional (3-D) scaffold culture systems were constituted, which would provide benefits for nutrition supply and metabolism during cell growth. Porosity and permeability of the 3-D alginate scaffolds were enhanced by using LIPUS with varied acoustic pressures, and the mechanism involved in the processes was investigated in detail. The porosity and permeability of the 3-D alginate scaffolds were quantitatively evaluated based on the scanning electron microscopy examination, in vivo fluorescence image observation, and laser con-focal image observation. The results indicated that the microstreaming generated by ultrasound-driven microbubble oscillations enhanced the porosity and permeability of the scaffolds, and the enhancement effect were increased with the increasing LIPUS driving pressures. In addition, the cell proliferation assessments verified that, HeLa cell grew better in the treated 3-D alginate scaffolds at an appropriate LIPUS pressure (e.g., 0.055 MPa), because the improved scaffold porosity and permeability could provide more cell growth space and benefit the nutrition supply.