The appropriate porosity and pore size of barrier membranes were associated with the transportation of biomolecules required for new bone formation and angiogenesis. In this study, we fabricated three-dimensional (3D)-printed resorbable polycaprolactone (PCL) membranes with different porosities (30%, 50%, and 70%) to evaluate the effective pore size for guided bone regeneration (GBR) membranes. To analyze mechanical properties and cytocompatibility, PCL membranes prepared using extrusion-based 3D printing technology were compared in dry and wet conditions and tested in vitro. The proliferation rates and pattern of fibroblasts and preosteoblasts on PCL membranes with different porosities were determined using a cell counting kit-8 assay and scanning electron microscopy. PCL membrane porosity did not affect cell proliferation, but osteogenic differentiation and mechanical properties were increased with lower porosity (30%) on day 14 (p < 0.001). Similar results were found in an in vivo calvarial defect model; new bone formation was significantly higher in PCL membranes with lower porosity (p < 0.001). These results indicate that 3D-printed PCL with 30% porosity (130 μm pore size) is an excellent pore size for GBR membranes.