Yersinia pestis, the causative agent of plague, has a feature of forming multicellular aggregates at liquid-air interface around the wall of glass tube. In this study, we employed the whole-genome DNA microarray of Y. pestis to investigate the global transcriptional profile in multicellularity compared with that in its planktonic growth. A total of 177 genes were differentially expressed in Y. pestis during early stage of multicellular formation; Seventy genes of them were up-regulated while 107 down-regulated. In addition to a large number of genes encoding unknown functions, most of the induced genes encode cell envelope and transport/binding proteins. The up-regulation of amino acid biosynthesis, the differentially altered genes that are involved in virulence, and the cold shock protein genes were for the first time reported to be associated with the multicellular formation. Our results revealed the global gene expression of Y. pestis were changed in the formation of multicellularity, providing insights into the molecular mechanism of multicellular behaviour, which need investigating further.