Host-microbe interactions in the intestine play a significant role in health and disease. Novel scaffolds for host cells, capable of potentially supporting these intricate interactions, are necessary to improve our current systems for mimicking host-microbiota interplay in vitro/ex vivo. In this research paper, we study the application of gelatin methacrylamide (GelMA) as scaffold material for intestinal epithelial cells in terms of permeability, mechanical strength, and biocompatibility. We investigated whether the degree of substitution (DS) of GelMA influences the permeability and found that both high and low DS GelMA show sufficient permeability of biorelevant transport markers. Additionally, we researched epithelial cell adherence and viability, as well as mechanical characteristics of different concentrations of GelMA. All concentrations of hydrogel show long-term biocompatibility for the mono- and co-cultures, despite the goblet-like cells (LS174T) showing lower performance than enterocyte-like cells (Caco-2). The mechanical strength of all hydrogel concentrations was in a physiologically relevant range to be used as scaffold material for intestinal cells. Lastly, we examined the effect of the two sterilization methods, ethylene oxide (EO) and 70% ethanol followed by UVC (EtOH/UVC). We found that the impact of the two methods on the mechanical characteristics was minimal, and we did not find a significant effect between the two methods on cell viability and confluency of Caco-2 cells seeded on the GelMA hydrogels. Based on these results, we conclude that GelMA is a suitable material as a scaffold for intestinal cell types in terms of permeability, mechanical strength and biocompatibility. These findings contribute to the growing field of in vitro modeling of the gut and moves the field further to ensuring more translatable research on host-microbe interactions.