Escherichia coli serotype O25b:H4 is involved in human urinary tract infections. In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient suffering from recurring UTI infections in an intensive care unit at Hera General Hospital in Makkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis and drug resistance of this isolate compared to other E. coli strains. We sequenced and analyzed the E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Using the ERGO genome analysis platform, we performed annotations and identified virulence and antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genome was assembled into four contigs representing a total chromosome size of 5.28 Mb, and three contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing the bla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to other uropathogenic E. coli genomes, we identified unique drug resistance and pathogenicity factors. In this work, whole-genome sequencing and targeted comparative analysis of a clinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strain encodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that are expressed constitutively in E. coli ST131. We identified the genes responsible for pathogenesis and drug resistance and performed comparative analyses of the virulence and antibiotic resistance determinants with those of other E. coli UPEC isolates. This is the first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.