Calreticulin is a calcium binding protein present primarily in the lumen of the endoplasmic reticulum. However, it can also localize to the cytoplasm adjacent to the cell membrane where it binds integrins, and to the nucleus. Recent studies showed that calreticulin inhibits DNA binding and transcriptional activity of glucocorticoid, androgen and retinoic acid receptors. The DNA binding domains of nuclear receptors share a common motif based upon the amino acid sequence KVFFKR which has been implicated in the binding of calreticulin. The vitamin D receptor (VDR) DNA binding domain contains the related motif KgFFrR. Here we show that calreticulin blocks specific DNA binding by the isolated VDR DNA binding domain in DNA mobility shift assays. Importantly, calreticulin blocks specific DNA binding by the full length VDR-RXR heterodimers. By contrast, calreticulin had no effect on specific DNA binding by the transcription factor ATF-a delta which lacks a KVFFKR-like motif in its DNA binding domain. We further showed that overexpression of calreticulin in the rat osteoblast-like cell line (ROS 17/2.8) inhibited the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] responsive transcriptional activation of a vitamin D-sensitive reporter gene, whereas the response to forskolin stimulation of a control promoter-reporter construct containing a cAMP response element (CRE), but no vitamin D response element (VDRE), was not affected by overexpression of calreticulin. Thus, calreticulin inhibits transcriptional activation by the VDR in vivo. Given the ubiquitous expression of calreticulin and the widespread expression of the VDR the studies described here may point to an important new mechanism whereby VDR mediated gene transcription can be modulated.