This study presents a novel 3D-printed bilayer vascular graft with gradient structures designed to promote organized vascular regeneration. The graft mimics the structure and mechanical properties of natural arteries, with an inner layer that has larger pores and high porosity to facilitate cell infiltration and alignment, and an outer layer with smaller pores and denser fiber intersections to provide mechanical strength. After one month of in vivo implantation, the 3D-printed grafts exhibited better pulsatility and compliance (8.9%) compared to electrospun grafts (1.9%), closely approaching that of natural arteries (11.36%). The 3D-printed grafts showed a three-layer structure resembling natural arteries, with complete reendothelialization and mature smooth muscle layer (SML) regeneration. In contrast, electrospun grafts showed incomplete endothelium and immature SML. The study highlights the importance of SML reconstruction in vascular graft regeneration and demonstrates that 3D-printed structures can rapidly promote vascular regeneration. The grafts also showed improved mechanical properties, with the 3D-printed graft having a bursting pressure of 5001 mmHg, which is sufficient for physiological blood vessel pressures. The results indicate that the 3D-printed grafts have a more favorable structure for vascular regeneration, with enhanced cell infiltration, tissue remodeling, and ECM remodeling. The study also shows that the 3D-printed grafts promote M2 macrophage polarization, which is associated with wound healing and vascular regeneration, while electrospun grafts promote M1 macrophage polarization, which is associated with inflammation and negative vascular regeneration. The study provides an effective strategy for vascular graft regeneration through 3D-printed structures.This study presents a novel 3D-printed bilayer vascular graft with gradient structures designed to promote organized vascular regeneration. The graft mimics the structure and mechanical properties of natural arteries, with an inner layer that has larger pores and high porosity to facilitate cell infiltration and alignment, and an outer layer with smaller pores and denser fiber intersections to provide mechanical strength. After one month of in vivo implantation, the 3D-printed grafts exhibited better pulsatility and compliance (8.9%) compared to electrospun grafts (1.9%), closely approaching that of natural arteries (11.36%). The 3D-printed grafts showed a three-layer structure resembling natural arteries, with complete reendothelialization and mature smooth muscle layer (SML) regeneration. In contrast, electrospun grafts showed incomplete endothelium and immature SML. The study highlights the importance of SML reconstruction in vascular graft regeneration and demonstrates that 3D-printed structures can rapidly promote vascular regeneration. The grafts also showed improved mechanical properties, with the 3D-printed graft having a bursting pressure of 5001 mmHg, which is sufficient for physiological blood vessel pressures. The results indicate that the 3D-printed grafts have a more favorable structure for vascular regeneration, with enhanced cell infiltration, tissue remodeling, and ECM remodeling. The study also shows that the 3D-printed grafts promote M2 macrophage polarization, which is associated with wound healing and vascular regeneration, while electrospun grafts promote M1 macrophage polarization, which is associated with inflammation and negative vascular regeneration. The study provides an effective strategy for vascular graft regeneration through 3D-printed structures.