The aim of this study was to fabricate bioresorbable polylactide (PLA) membranes by 3D printing and compare their properties to those of the membranes fabricated by the conventional method and compare the effect of different pore sizes on the properties of the 3D-printed membranes. PLA membranes with three different pore sizes (large pore-479 μm, small pore-273 μm, and no pore) were 3D printed, and membranes fabricated using the conventional solvent casting method were used as the control group. Scanning electron microscopy (SEM) and micro-computed tomography (µ-CT) were taken to observe the morphology and obtain the porosity of the four groups. A tensile test was performed to compare the tensile strength, elastic modulus, and elongation at break of the membranes. Preosteoblast cells were cultured on the membranes for 1, 3 and 7 days, followed by a WST assay and SEM, to examine the cell proliferation on different groups. As a result, the 3D-printed membranes showed superior mechanical properties to those of the solvent cast membranes, and the 3D-printed membranes exhibited different advantageous mechanical properties depending on the different pore sizes. The various fabrication methods and pore sizes did not have significantly different effects on cell growth. It is proven that 3D printing is a promising method for the fabrication of customized barrier membranes used in GBR/GTR.