This study investigates if the application of bone marrow-derived mesenchymal stem cells (BM-MSCs) loaded 3D-printed scaffolds could improve rotator cuff repair. The polylactic-co-glycolic acid (PLGA) scaffolds were fabricated by 3D print technology. Rabbit BM-MSCs were transfected with a recombinant adenovirus encoding bone morphogenic protein 12 (BMP-12). The effect of BM-MSCs loaded PLGA scaffolds on tendon-bone healing was assessed by biomechanical testing and histological analysis in a rabbit rotator cuff repair model. We found that the PLGA scaffolds had good biocompatible and biodegradable property. Overexpression of BMP-12 increased the mRNA and protein expression of tenogenic genes in BM-MSCs cultured with DMEM medium and seeded in PLGA scaffolds. When BMP-12-overexpressing BM-MSCs-loaded PLGA scaffolds were implanted into the injured rabbit supraspinatus tendon-bone junctions, the tendon-bone healing was improved. Our results suggest that application of BMP-12 overexpressing BM-MSCs loaded 3D-printed PLGA scaffolds promote the healing of tendon-bone interface, improve collagen organization and increase fibrocartilage in the rabbit rotor cuff repair. Rotator cuff regeneration achieved by BMP-12-overexpressing BM-MSCs-loaded PLGA scaffolds may represent a novel approach for the management of rotator cuff defect.