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Background/Objectives: Bioprinting is an innovative technology in tissue engineering, enabling the creation of complex biological structures. This study aims to develop a three-dimensional (3D) bioprinted model of Müller cells (MCs) to enhance our understanding of their physiological and pathological roles in the retina. Methods: We investigated two different hydrogels for their ability to support the viability and differentiation of rMC-1 cells, an immortalized retinal cell line. Using 3D bioprinting technology, we assessed cell viability, differentiation, and functional characteristics through various assays, including live/dead assays and western blot analysis. Results: The collagen-based hydrogel significantly improved the viability of rMC-1 cells and facilitated the formation of spheroid aggregates, more accurately mimicking in vivo conditions compared to traditional two-dimensional (2D) culture systems. Moreover, 3D bioprinted MCs exhibited reduced markers of gliosis and oxidative stress compared to 2D cultures. Molecular analysis revealed decreased expression of GFAP and phosphorylated ERK in the 3D setting, indicating a less stressed cellular phenotype. Conclusions: Our findings demonstrate that 3D bioprinting technologies provide a more predictive platform for studying the biology of retinal MCs, which can help in the development of targeted therapeutic strategies for retinal diseases. [ABSTRACT FROM AUTHOR]