In the current study, we apply nuclear proteome analysis to the search for novel nuclear proteins that play evolutionarily conserved roles in the sugar response in plants. We initially identified 563 different proteins by nanoLC/ESI/MS/MS analysis of extracts from rice nuclei that were purified by Percoll density gradient centrifugation, whereas 307 different proteins were identified with nucleic acid-associated proteins that were enriched by DNA affinity chromatography. Since some proteins were identified in both samples, the identified proteins summed up to 657 different proteins. Among them, we found not only transcription and splicing factors, but also a mediator of sugar signaling in plants, hexokinase. For identification of evolutionarily conserved and sugar-responsive nuclear factors, we performed in silico screening that included the identification of Arabidopsis homologues of our panel of rice proteins and investigation of the sugar-responsiveness of the corresponding genes. Since two WD40-like proteins and an armadillo/pumilio-like protein were selected as candidates for such nuclear factors, we confirmed the nuclear localization of these proteins and sugar-induced expression of their corresponding genes in both Arabidopsis and rice. We thus improve our knowledge of plant nuclear proteins and present a novel approach to the identification of factors with particular characteristics.