Lines of evidence indicate that nuclear pore complex profoundly affects the timing of plant flowering, but the underlying mechanisms remain poorly elucidated. Here, we report that Nucleoporin 96 (Nup96) acts as a negative regulator of long-day photoperiodic flowering in Arabidopsis. Through multiple approaches, we identified and verified an E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) interacting in vivo with Nup96. Nup96 and HOS1 proteins mainly localize and interact on nuclear membrane. Loss-of-function of Nup96 leads to destruction of HOS1 proteins without changing its mRNA abundance, which results in over-accumulation of the key activator of long-day photoperiodic flowering, CONSTANS (CO) proteins, as that in hos1 mutants. Unexpectedly, we observe that mutation of HOS1 gene strikingly diminishes Nup96 protein level, suggesting that Nup96 and HOS1 are mutually stabilized and thus they form a novel repressive module to regulate CO protein turnover. Therefore, the nup96 and hos1 single and nup96 hos1 double mutants have very much similar early-flowering phenotypes and overlapping transcriptome changes. Together, this study reveals a new repression mechanism, where the Nup96-HOS1 repressive module gates the level of CO proteins, to prevent precocious flowering of Arabidopsis in long-day conditions.,© 2019 American Society of Plant Biologists. All rights reserved.