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3D microstructured biomaterials are favorable in tissue engineering due to their superior guidance to cellular activities. Herein, we developed a 3D microstructured bacterial cellulose (BC) with arranged fibers by controlling Acetobacter xylinum through an electric field (EF) application. The real-time video analysis showed that EF directed the migration of A. xylinum and increased its migration speed with the increased EF. The bacteria quickly changed direction with high motility in response to the switch on/off of the EF. In the long-term EF stimulation (ES), bacterial cells were oriented along the direction of EF while bacteria simultaneously produced nanocellulose, resulting in three-dimensional (3D) networks with aligned fibers. Moreover, the prepared 5 mA-BC hydrogels presented the ordered 3D microstructure with significantly higher fiber alignment and diameter than the NO EF-BC hydrogels. The in vitro biological evaluation demonstrated that the 5 mA-BC hydrogels were biocompatible whereon NIH3T3 cells proliferated along the direction of fiber alignment. These findings demonstrate that ES provides a promising strategy for the natural fabrication of aligned 3D microstructured BC to guide cellular activities for tissue engineering.