We report the identification and characterization of cellulose as an extracellular matrix component present in the biofilm of a E. sakazakii clinical isolate, after growth in nutrient deficient (M9) media. Using a bacterial artificial cloning (BAC) approach in E. coli, with subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. Among the genes already described for cellulose production in E. coli two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass cover slips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.