Biological macromolecular machines perform impressive mechanical movements. F‐adenosine triphosphate (ATP) synthase uses a proton gradient to generate ATP through mechanical rotations. Here, a programmed hexagonal DNA nanomachine, in which a three‐armed DNA nanostructure (TAN) can perform stepwise rotations in the confined nanospace powered by DNA fuels, is demonstrated. The movement of TAN can precisely go through a 60° rotation, which is confirmed by atomic force microscopy, and each stepwise directional rotating is monitored by fluorescent measurements. Moreover, the rotary nanomachine is used to spatially organize cascade enzymes: glucose oxidase (GOx) and horseradish peroxidase (HRP) in four different arrangements. The multistep regulations of the biocatalytic activities are achieved by employing TAN rotations. This work presents a new prototype of rotary nanodevice with both angular and directional control, and provides a nanoscale mechanical engineering platform for the reactive molecular components, demonstrating that DNA‐based framework may have significant roles in futuristic nanofactory construction.