The permeability-based assay is commonly used to assess intestinal barrier function, and it relies on using a transwell insert as an essential compartment. The device consists of a semipermeable membrane that is attached at the bottom of the insert and splits the system into the apical and basolateral compartments. However, commercial inserts are standardized with different pore sizes based on the application and offer only a flat plane of two-dimensional cell culture. Herein, we present a simple, low-cost 3D-printed transwell device and a robust method to functionalize the inserts for paper-based 3D cell culture. This 3D-printed device was fabricated from a polylactic acid (PLA) filament, and a paper membrane used to support HT-29 cells for intestinal permeability assessment. A device showed good biocompatibility when culturing HT-29 cells for 48 and 72 h with 97 % and 98 % cell viability, respectively. Together with fluorescence images, cells were attached directly to the microfiber networks of a Matrigel-functionalized paper, indicating that the functionalized paper is biocompatible and bioactive. Furthermore, in a more appropriate culture microenvironment, SEM analyses revealed cellular features differentiating into mucus-secreting cells, evidenced by the formation of microvilli on the cell surface, which was further confirmed by immunofluorescence staining of villin-1. To demonstrate the usability of the 3D-printed transwell device, intestinal permeability was assessed using both chemical and biological stimulation treatments. The permeability results employing FITC-dextran validated the association between a different level of relative fluorescence intensity unit (RFU) and the orange color of live cells by CellTrackerTM. As a result, this 3D-printed transwell device provides a straightforward and cost-effective method for manufacturing a device for customization in many laboratory settings, making it a feasible alternative to marketed transwell devices that do not allow for customization.