A 43-bp distal element, the AtKP1-related element (KPRE), was previously shown to repress the promoter activity of the kinesin gene AtKP1 in Arabidopsis thaliana. In order to identify KPRE-binding factor 1 (KBF1), a combination of ion-exchange chromatography, gel-filtration chromatography and DNA-affinity chromatography was used to purify KBF1 from whole cell extracts of Arabidopsis seedlings. Mass spectrometric identification showed that KBF1 contains two members of the whirly family of transcription factors, AtWHY1 and AtWHY3. KBF1 is a single and double-stranded DNA-binding factor. A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. Over-expression of AtWHY1 and AtWHY3 led to an obvious decrease of AtKP1 transcripts, based on quantitative real-time PCR analysis. Interestingly, salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts, and a decrease of AtKP1 transcripts. Thus, AtWHY1 and AtWHY3, as two components of KBF1, can be recruited at the KPRE site to mediate the transcriptional repression of AtKP1. Our results prove that AtKP1 is a new downstream target of the whirly family of transcription factors.