Novel additive manufacturing processes are increasingly recognized as ideal techniques to produce 3D biodegradable structures with optimal pore size and spatial distribution, providing an adequate mechanical support for tissue regeneration while shaping in-growing tissues. With regard to the mechanical and biological performances of 3D scaffolds, pore size and geometry play a crucial role. In this study, a novel integrated automated system for the production and in vitro culture of 3D constructs, known as BioCell Printing, was used only to manufacture poly(ε-caprolactone) scaffolds for tissue engineering; the influence of pore size and shape on their mechanical and biological performances was investigated. Imposing a single lay-down pattern of 0◦/90◦ and varying the filament distance, it was possible to produce scaffolds with square interconnected pores with channel sizes falling in the range of 245–433 μm, porosity 49– 57% and a constant road width. Three different lay-down patterns were also adopted (0◦/90◦, 0◦/60/120◦ and 0◦/45◦/90◦/135◦), thus resulting in scaffolds with quadrangular, triangular and complex internal geometries, respectively. Mechanical compression tests revealed a decrease of scaffold stiffness with the increasing porosity and number of deposition angles (from 0◦/90◦ to 0◦/45◦/90◦/135◦). Results from biological analysis, carried out using human mesenchymal stem cells, suggest a strong influence of pore size and geometry on cell viability. On the other hand, after 21 days of in vitro static culture, it was not possible to detect any significant variation in terms of cell morphology promoted by scaffold topology. As a first systematic analysis, the obtained results clearly demonstrate the potential of the BioCell Printing process to produce 3D scaffolds with reproducible well organized architectures and tailored mechanical properties. (Some figures may appear in colour only in the online journal)