This study presents a method for fabricating living, heterogeneous aortic valve conduits using 3D bioprinting with alginate/gelatin hydrogels. The research demonstrates the ability to create anatomically complex structures with cellular heterogeneity, incorporating two distinct cell types—smooth muscle cells (SMC) and valve interstitial cells (VIC)—in specific regions. The hydrogels were printed with direct encapsulation of SMC in the valve root and VIC in the leaflets, resulting in viable cells with high survival rates (81.4±3.4% for SMC and 83.2±4.0% for VIC) after 7 days in culture. Encapsulated SMC expressed elevated alpha-smooth muscle actin in stiff matrices, while VIC expressed elevated vimentin in soft matrices, indicating distinct phenotypic responses. The hydrogels exhibited reduced mechanical properties over time, but cell-laden hydrogels maintained their biomechanical integrity. The study highlights the potential of 3D bioprinting for creating living, functional aortic valve conduits with the ability to remodel, regenerate, and grow, offering a promising alternative to traditional prosthetic valves. The results suggest that 3D bioprinting can fabricate clinically relevant, heterogeneous aortic valve conduits with anatomical complexity and cellular diversity, which could be used for tissue-engineered valve replacements. The study also discusses the challenges of current fabrication methods and the advantages of 3D bioprinting in achieving complex, cell-encapsulated structures with tailored biomechanical properties. The research provides a foundation for future developments in tissue engineering and regenerative medicine, particularly in the field of heart valve replacement.This study presents a method for fabricating living, heterogeneous aortic valve conduits using 3D bioprinting with alginate/gelatin hydrogels. The research demonstrates the ability to create anatomically complex structures with cellular heterogeneity, incorporating two distinct cell types—smooth muscle cells (SMC) and valve interstitial cells (VIC)—in specific regions. The hydrogels were printed with direct encapsulation of SMC in the valve root and VIC in the leaflets, resulting in viable cells with high survival rates (81.4±3.4% for SMC and 83.2±4.0% for VIC) after 7 days in culture. Encapsulated SMC expressed elevated alpha-smooth muscle actin in stiff matrices, while VIC expressed elevated vimentin in soft matrices, indicating distinct phenotypic responses. The hydrogels exhibited reduced mechanical properties over time, but cell-laden hydrogels maintained their biomechanical integrity. The study highlights the potential of 3D bioprinting for creating living, functional aortic valve conduits with the ability to remodel, regenerate, and grow, offering a promising alternative to traditional prosthetic valves. The results suggest that 3D bioprinting can fabricate clinically relevant, heterogeneous aortic valve conduits with anatomical complexity and cellular diversity, which could be used for tissue-engineered valve replacements. The study also discusses the challenges of current fabrication methods and the advantages of 3D bioprinting in achieving complex, cell-encapsulated structures with tailored biomechanical properties. The research provides a foundation for future developments in tissue engineering and regenerative medicine, particularly in the field of heart valve replacement.