Tubular structures contribute to essential organ functions. Therefore, controlling tubulogenesis is essential for bottom-up tissue engineering approaches. Tissue engineering strategies to form tubular structures utilize extracellular matrix (ECM) components and micropatterned molds. To improve the tubular formation rate, we studied the substrate stiffness’s influence on the tubulogenesis of murine inner medullary collecting duct (mIMCD) cells. mIMCD cells were seeded in micropatterned molds with different compositions of polydimethylsiloxane (PDMS) (1:5, 1:10, 1:15, 1:20, 1:30) and agarose (1%, 2%, 3% 5%). We established the Young’s modulus of the PDMS and agarose substrates and determined the ideal substrate stiffness for tube formation to be between 277 kPa and 2610 kPa. Within our parameters, optimal tube formation was observed at 439.9 kPa, a value similar to the Young’s Modulus found in the basement membrane of the murine renal tubular compartment. We also found that different substrate concentrations of agarose or PDMS are associated with different expression levels of the apical polarization marker Zonula occludens 1 (ZO-1) in the generated tubular structures. In addition to the substrate stiffness, we observed that the tube formation differed based on the substrate material, with agarose showing a generally greater tube formation rate. While previous research demonstrated that ECM stiffness influences cellular behavior towards tube formation, our results suggest that the stiffness of the substrate influences tubular formation independently of the ECM.