Bacterial cellulose, as a kind of natural biopolymer produced by bacterial fermentation, has attracted wide attention owing its unique physical and chemical properties. Nevertheless, the single functional group on the surface of BC greatly hinders its wider application. The functionalization of BC is of great significance to broaden the application of BC. In this work, N-acetylated bacterial cellulose (ABC) was successfully prepared using K. nataicola RZS01-based direct synthetic method. FT-IR, NMR and XPS confirmed the in-situ modification of BC by acetylation. The SEM and XRD results demonstrated that ABC has a lower crystallinity and higher fiber width compare with pristine 88 BCE % cell viability on NIH-3 T3 cell and near zero hemolysis ratio indicate its good biocompatibility. In addition, the as-prepared acetyl amine modified BC was further treated by nitrifying bacteria to enrich its functionalized diversity. This study provides a mild in-situ pathway to construct BC derivatives in an environmentally friendly way during its metabolism.