Cells respond to changes in environmental conditions via orchestrated modifications in gene expression. For example, in response to heat shock, cells execute a program of gene-specific transcriptional activation and repression. Although the activation of genes upon heat shock has been widely studied, the mechanism of mRNA transcriptional repression upon heat shock is unexplained. Here we show that during the heat shock response in mouse cells, a small noncoding RNA polymerase III transcript, B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator and reveal how mRNA transcription is repressed upon heat shock. Moreover, we identify a function for B2 RNA, which is transcribed from short interspersed elements that are abundant in the mouse genome and historically considered to be 'junk DNA.'