(1) Autologous chondrocyte implantation (ACI) is a prominent method for treating cartilage damage, but periosteal patches can cause chondrocyte leakage. This study evaluates the potential of a decellularized membrane derived from the cell-produced extracellular matrix of 1-day-old porcine cartilage (pcECM-DM) to act as a substitute for periosteal patches. (2) The interaction between young rabbit chondrocyte cells and pcECM-DM was assessed through cytotoxicity, differentiation, cell viability, cell migration, and adhesive ability. Rabbit chondrocyte cells, cultivated until passage two, were seeded onto a 6 mm diameter membrane. Assessments included DAPI-PKH26 staining, histological staining, live/dead assay, WST-1 assay, and proteomics analysis. (3) Results: DAPI-PKH26 staining showed successful adhesion and the uniform distribution of cells on the membrane. Safranin-O and H&E staining confirmed that the membrane supports chondrocyte adhesion and extracellular matrix production with high cell density and typical chondrocyte morphology. The live/dead assay demonstrated a high proportion of viable cells at 24 and 48 h, with increased cell proliferation over time. The WST-1 assay showed a significant increase in OD450 values, confirming cell proliferation and biocompatibility. Proteomic analysis revealed the significant enrichment of genes associated with extracellular matrix organization, cell adhesion, and cartilage development. (4) Conclusions: This novel biomaterial holds the potential to enhance cartilage regeneration and offer a viable alternative to periosteal patches.