Many gut bacteria degrade polysaccharides, providing nutritional advantages to their hosts. Fucose, a mucin degradation product, was suggested as a communication molecule between the resident microbiota and external pathogens. However, the precise role and variants of the fucose utilization pathway remain to be elucidated. Here, we computationally and experimentally investigated the fucose utilization operon ofE. coli. While the operon is conserved amongE. coligenomes, a variant pathway, in which an ABC transporter system replaces the fucose permease gene (fucP), was computationally identified in 50 out of 1058 genomes. Comparative genomics and subsystems analysis results were confirmed by polymerase chain reaction-based screening of 40 humanE. coliisolates, which indicated the conservation offucPin 92.5% of the isolates (vs. 7.5% of its suggested alternative,yjfF). The in silico predictions were confirmed by in vitro experiments comparing the growth ofE. colistrains K12, BL21, and isogenic fucose-utilization K12 mutants. Additionally,fucPandfucItranscripts were quantified inE. coliK12 and BL21, after in silico analysis of their expression in 483 public transcriptomes. In conclusion,E. coliutilizes fucose by two pathway variants, with measurable transcriptional differences. Future studies will explore this variation’s impact on signaling and virulence.