Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O -fucosyltransferase SPINDLY (SPY) and the O -GlcNAc transferase SECRET AGENT (SEC) are two prominent O -glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O -GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDORESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPYinteracting protein. PRR5 can be O -fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O -fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O -fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O -fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O -GlcNAcylation in mammals, but via O -fucosylation in higher plants.