Cartilage defects negatively impact the quality of life of over 500 million people worldwide. 3D-printed scaffolds loaded with polydatin (PD) have shown significant potential in cartilage defect repair. This study aims to investigate their reparative effects and, using lipidomics techniques, to analyze the related metabolic changes, thereby providing new strategies for treating cartilage defects. Biocompatible 3D-printed scaffolds containing PD were prepared. Thirty New Zealand rabbits were divided into three groups: Normal group, Model group, and Scaffold group, with 10 rabbits in each group. After three months of intervention, the repair of cartilage defects was evaluated through macroscopic observation, micro-CT, H&E staining, and Safranin O/fast green (SFO/FG) staining. The expression of VEGFA, Col2a1, and Biglycan was detected by immunofluorescence. Additionally, newly formed cartilage tissue was collected for lipid metabolic profiling analysis to comprehensively evaluate the mechanism of action of the PD-loaded 3D-printed scaffold in cartilage repair. Macroscopic observation, micro-CT, H&E staining, and SFO/FG staining indicated that the repair of cartilage defects in the Scaffold group was significantly better than in the Model group, approaching the level of the Normal group. Lipidomics revealed that the Scaffold group modulated 36 metabolites, with a modulation rate of 69.23%. The main modulated metabolites included ceramides (Cers), glycerophospholipids (GPs) and sphingomyelins (SMs). Furthermore, immunofluorescence analysis showed increased expression of cartilage cell markers Sox9, Col2a1, Biglycan, and VEGFA, along with a reduction in cell apoptosis (all p < 0.05) after scaffold implantation. The 3D-printed scaffold loaded with PD may promote cartilage repair by correcting the disorder of lipid metabolites in cartilage tissue, inhibiting chondrocyte apoptosis, enhancing the expression of vascular-related factors, and accelerating the remodeling of cartilage collagen matrix.