The small ventral lateral neurons (sLNvs) are the main clock neurons in fruitflies and show circadian changes in excitability and axonal terminal width, which are at their greatest at dawn. This study revealed that sLNvs also show circadian changes in axon volume, suggesting the involvement of an actin-related mechanism. Indeed, levels of Rho1 — a GTPase implicated in actin dynamics — exhibited circadian oscillations in sLNvs, and overexpressed Rho1 maintained axon terminals in a retracted state, leading to arrhythmic circadian behaviour and impaired regulation of downstream clock neurons. Thus, oscillatory Rho1 activity is a key circadian regulator in fruitflies.