Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections, for which antibiotic therapies often fail. UPEC forms biofilms during infection, leading to enhanced protection from antibiotics and host immune factors. Cellulose is a polysaccharide produced by UPEC that acts as an extracellular glue to stabilize the biofilm. Cellulose production is regulated by a complex pathway involving the second messenger cyclic-di-GMP and the curli positive regulator CsgD. In this study, we employed a combination of transposon mutagenesis and transposon-directed insertion site sequencing (TraDIS), together with a phenotypic screen to detect cellulose, to identify the set of genes required for cellulose production. The involvement of these genes in cellulose production was validated by constructing defined mutants and subsequent complementation. In total, 14 genes were identified that had a significant contribution to cellulose production. This included 5/9 genes from the co-located divergent cellulose biosynthesis operons – bcsQ (secretion component), bcsA (cellulose synthase catalytic subunit), bcsB (cellulose synthase membrane anchor), bcsC (porin) and bcsG (phosphoethanolamine transferase). A diguanylate cyclase gene (dgcC) that synthesizes cyclic-di-GMP, as well as genes involved in metabolism (pgi, pgm and fbp), purine de novo biosynthesis (purD and purH) and sodium transport (nhaA) were also identified. New genes involved in cellulose biosynthesis were also identified and validated, comprising genes involved in lipid-A core biosynthesis (waaD, waaC and waaG) and ATP synthesis (atpA and atpB). Finally, we also identified a unidirectional Tn5 insertion upstream of the pdeH gene (encoding a phosphodiesterase that degrades cyclic-di-GMP) and showed that strong expression of PdeH induced by plasmid over-expression led to loss of cellulose production. Overall, this study has revealed a complex network of genes involved in cellulose regulation and biosynthesis in UPEC, providing new insight to unravel the contribution of this bacterial polysaccharide in biofilm formation.