AbstractIn Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c‐di‐GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type‐I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type‐I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c‐di‐GMP‐regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12‐gene cluster, Bcam1330–Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow‐chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330–Bcam1341 cluster and that this binding is enhanced by the presence of c‐di‐GMP. Furthermore, we demonstrate that overproduction of both c‐di‐GMP and Bcam1349 leads to increased transcription of these genes, indicating that c‐di‐GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330–Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330–Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c‐di‐GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.