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Mechanically twisting a yarn is a useful and important method for creating internal stress, changing macromolecular orientation, and affording interesting fiber properties, including mechanical, structural, physical and chemical properties. Bacterial cellulose (BC) nanofibers are a new type of highly crystalline bionanofibers exhibiting excellent intrinsic mechanical properties. Herein, BC macrofibers with a diameter of 0.35–0.53 mm were prepared from BC hydrogel films using a two-step technique involving drying and twisting processes. Moreover, the effects of the drying method (oven- and freeze-drying) and those of the processing steps on the morphologies, structures, and physicochemical properties of the macrofibers were investigated. Results indicated that only the wet twisting–first process affords continuous macrofibers with a compact and uniform structure. The tensile strength and elongation at break of the macrofibers were 199 MPa and 24%, respectively. Furthermore, by introducing graphene oxide (GO) to the macrofibers, a photothermal and moisture actuator was facilely constructed, which could produce a twisting motion of 0.39 rpm and an untwisting motion of 0.6 rpm. This study provides insights into methods to easily alter the properties of BC macrofibers, which is pivotal for the macrofiber's potential application in intelligent devices. [Display omitted] • The surface of T-OD macrofibers is smooth and flat, and the tensile stress can reach 199 MPa. • The GO-T-OD macrofiber can produce reversible motion under moisture and NIR light stimulation, and the rotation angle can be increased to 121°/cm in 25 s • Reversible twisting artificial muscles offer unlimited possibilities for future applications in artificial muscles and other intelligent drives. [ABSTRACT FROM AUTHOR]