Escherichia coli (E. coli) is one of the most common uropathogenic bacteria. the emergence of multidrug resistance among these bacteria resulted in a worldwide public health problem which requires alternative treatment approaches such as phage therapy. in this study, phage VB_ecoS-Golestan, a member of Siphoviridae family, with high lytic ability against E. coli isolates, was isolated from wastewater. Its burst size was large and about 100 plaque-forming units/infected cell, rapid adsorption time, and high resistance to a broad range of pH and temperatures. Bioinformatics analysis of the genomic sequence suggests that VB_ecoS-Golestan is a new phage closely related to Escherichia phages in the Kagunavirus genus, Guernseyvirinae subfamily of Siphoviridae. the genome size was 44829 bp bp that encodes 78 putative ORFs, no tRNAs, 7 potential promoter sequences and 13 Rho-factor-independent terminators. no lysogenic mediated genes were detected in VB_ecoS-Golestan genome. overall VB_ecoS-Golestan might be used as a potential treatment approach for controlling E. coli mediated urinary tract infection, however, further studies are essential to ensure its safety. E. coli is one of the most common pathogens causing urinary tract infection.Uropathogenic strains of Escherichia coli (UPECs) account for 70-95% of community-acquired UTIs and over 50% of nosocomial UTIs 1-4. UPECs expresses several virulence factors to break the inertia of the mucosal barrier. An important virulence factor of these bacteria is their ability to adhere to the host epithelial cells, which is a precondition for the creation of infectious diseases, mostly through the expression of fimbriae 5,6. Emergence of antibiotic resistance among UPEC strains can occur through several mechanisms, and has been reported frequently over the last decade. This made them a worldwide concern as it significantly reduced the efficiency of the first-line treatment agents (like ampicillin, nitrofurantoin, sulfamethoxazole/trimethoprim (SXT) and fluoroquinolones) against this pathogen 7. Emergences of such multi-drug resistant (MDR) strains limits the treatment options 3,8-11. Therefore, finding new effective strategies for treatment of MDR UPECs is a must 7. Nowadays, phage therapy, once more, has become a hopeful biological method to manage antibiotic-resistant infections. This is as a result of the increased knowledge on biology of phage, very high specificity toward their host bacteria, no effect on the normal microflora, and replication and exponential growth in presence of their specific hosts at the infection site 12,13. Since phage-therapy is still an understudy therapeutic approach, further development of this method requires biological characterization of bacteriophages such as their host specificity, genome diversity, and adaption to their bacterial hosts 14,15. The current study aimed for comprehensive biological and genomic characterizations of a new lytic bacteriophage against uropathogenic E. coli.