We investigated the autonomous bottom-up fabrication of three-dimensional honeycomb cellulose structures, using Gluconacetobacter xylinus as a bacterial nanoengine, on cellulose honeycomb templates prepared by casting water-in-oil emulsions on glass substrates (Kasai and Kondo, Macromol. Biosci., 4, 17-21, 2004). The template film had a unique molecular orientation state along the honeycomb frames, but was non-crystalline. When G. xylinus, used as a nanofiber-producing bacterium, was incubated on the honeycomb scaffold in a culture medium, it secreted cellulose nanofibers only on the upper surface of the honeycomb frame. The movement was regulated by a selective interaction between the synthesized nanofiber and the surface of the honeycomb frames of the template. The relationship between directed deposition of synthesized nanofibers and ordered fabrication from the nano- to the micro-scale could provide a novel bottom-up methodology, using bacteria, for the design of three-dimensional honeycomb structures as functional materials with nano/micro hierarchical structures, with low energy consumption. (c) 2014, The Society for Biotechnology, Japan. All rights reserved.