Technical Abstract: Shiga-toxigenic Escherichia coli O157:H7 (STEC) outbreaks have been linked to consumption of fresh produce. Cellular appendages, such as curli fibers have been suggested to be involved in STEC persistence in fresh produce as these curli are critical in biofilm formation and adherence to animal cells. We determined the role of curli and cellulose in attachment of STEC on spinach leaves. Six STEC strains: wild, curli-deficient (delta csgA, delta cgsD) cellulose deficient (delta bcsA), and curli + cellulose deficient (delta csgA/delta bcsA) mutants were evaluated for curli and cellulose expression. Spinach leaves were spot-inoculated 7 log CFU/leaf bacteria and incubated at 22ºC for 48 h. Populations of loosely and strongly attached E. coli O157:H7 on spinach leaves were determined by spiral plating on Sorbitol MacConkey agar. Further, attachment was confirmed by Laser Scanning Confocal Microscopy (LSCM). STEC delta csgA and delta cgsD mutants didn’t express curli at 22ºC, whereas wild type and curli-restored strains developed a clear red phenotype on CR. SEM and LSCM analysis confirmed cells expressing extracellular matrix containing curli fibers. Curli-expressing STEC strains strongly attached to spinach leaves at significantly higher numbers than that of curli-deficient strains. Lack of cellulose expression did not affect stronger attachment of these strains to spinach leaves. LSCM analysis of inoculated spinach revealed that strongly attached curli-expressing cells were surrounded by an extracellular matrix. Understanding the role of curli in STEC attachment and persistence in vegetal matrices will help developing intervention strategies to remove pathogens from fresh produce and thereby reduce future recalls.