| Summary: | <b>: </b>Circadian clocks drive rhythmic physiology and behavior to allow adaption to daily environmental changes. In <i>Drosophila</i>, the small ventral lateral neurons (sLNvs) are primary pacemakers that control circadian rhythms. Circadian changes are observed in the dorsal axonal projections of the sLNvs, but their physiological importance and the underlying mechanism are unclear. Here, we identified <i>miR-263b</i> as an important regulator of circadian rhythms and structural plasticity of sLNvs in <i>Drosophila</i>. Depletion of <i>miR-263b</i> (<i>miR-263b</i><sup>KO</sup>) in flies dramatically impaired locomotor rhythms under constant darkness. Indeed, <i>miR-263b</i> is required for the structural plasticity of sLNvs. <i>miR-263b</i> regulates circadian rhythms through inhibition of expression of the LIM-only protein <i>Beadex</i><i> (Bx).</i> Consistently, overexpression of <i>Bx</i> or<i> </i>loss-of-function mutation (<i>Bx</i><sup>hdpR26</sup>) phenocopied <i>miR-263b</i><sup>KO</sup> and <i>miR-263b </i>overexpression in behavior and molecular characteristics. In addition, mutating the <i>miR-263b</i> binding sites in the <i>Bx</i> 3′ UTR using CRISPR/Cas9 recapitulated the circadian phenotypes of <i>miR-263b</i><sup>KO</sup> flies. Together, these results establish <i>miR-263b</i> as an important regulator of circadian locomotor behavior and structural plasticity.
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