The understanding about virulence factors (VFs) and the drug resistance of uropathogenic Escherichia coli (UPEC) helps us understand the pathogenesis of urinary tract infections (UTIs) and make better decisions for clinical treatment. This study examined the correlation between the extended-spectrum β-lactamases (ESBLs) phenotype and VFs in UPEC strains. In addition, we validated the therapeutic potential of fosfomycin in acute pyelonephritis mice. From May 2017 to November 2018, 22 nonduplicate E coli. strains were isolated from UTI patients. PCR was utilized to detect the distribution of virulence genes. We also analyzed the ESBL phenotype in E coli. We further evaluated the therapeutic effect of intravenous fosfomycin treatment in the acute pyelonephritis (APN) model. All 22 UPEC strains expressed the type 1 fimbriae (FimH) gene and more than 50% (12/22) of strains produced ESBLs. The detection rates of the iron acquisition-associated genes ChuT and IutA were 77.3% ( n = 17 ) and 50% ( n = 11 ) and those of P fimbria papA and papC genes were 45% ( n = 10 ) and 50% ( n = 11 ), respectively. Though the VFs were closely related with pathologenicity, the relationship between VFs and ESBLs still needs further investigation. Furthermore, intravenous fosfomycin 800 mg/kg significantly reduced the bacterial load and the inflammatory infiltration in the bladder and kidney, maintaining the structural integrity of the kidney. Intravenous fosfomycin administration can be used for the treatment of acute pyelonephritis caused by highly pathogenic and drug-resistant UPEC strains.