Bacterial nanocellulose (BNC) shows high biocompatibility as wound dressing or dura mater , blood vessel, and cartilage implant. Three-dimensional perforation (3-D-∅) favors migration of chondrocytes into the BNC and cartilage matrix formation. Thus, the regenerative capacity of 3-D-∅ BNC implants was tested in a standardized bovine cartilage punch model. Cartilage rings containing a central defect with an outer diameter of 6 mm and an inner diameter of 2 mm were prepared from the trochlear groove (femur-patellar articulation site). Three-D-∅ BNC implants (cell-free or cell-loaded) were cultured inside the cartilage rings for up to 12 weeks. Cartilage-BNC-constructs were then investigated by histology (hematoxylin/eosin; safranin O) and immunohistology (aggrecan, collagens 1 and 2), as well as for protein content, RNA expression, and implant push-out force. Cartilage-BNC-constructs remained vital with preserved matrix integrity during culture and almost no loss of matrix-bound proteoglycan (aggrecan) or collagen 2 from ‘host’ cartilage (with very limited quantities of collagen 1). Interestingly, 3-D-∅ BNC implants displayed: (1) significantly increased superficial, but also 3-D cell seeding over time (cell-loaded significantly earlier than cell-free); (2) progressively increased aggrecan/collagen 1 and collagen 2/collagen 1 mRNA ratios, as well as aggrecan and collagen 2 protein levels; and (3) significantly increased push-out forces over time (cell-loaded). Progressively increasing cell seeding and chondrogenic differentiation suggest beginning cartilage regeneration of the 3-D-∅ BNC in this model system, and indicate an excellent potential of 3-D-∅ BNC as a cartilage replacement material. Cell-loading may favor implant performance by accelerating cell colonization. Graphical abstract