| Crynodeb: | The emergence of multidrug-resistant bacterial pathogens has severely threatened global health. A phage with the ability to efficiently and specifically lyse bacteria is considered an alternative for controlling multidrug-resistant bacterial pathogens. The discovery of novel agents for controlling the infections caused by <i>K. pneumoniae</i> is urgent due to the broad multidrug-resistance of <i>K. pneumoniae</i>. Only a few phage isolates have been reported to infect multidrug-resistant <i>K. pneumoniae</i>. In this study, by using the multidrug-resistant <i>K. pneumoniae</i> strain as an indicator, a novel phage called vB_KleS-HSE3, which maintains high antibacterial activity and high physical stability, was isolated from hospital sewage. This phage infected one of four tested multidrug-resistant <i>K. pneumoniae</i> strains. This phage belongs to the Siphoviridae family and a comparative genomic analysis showed that this phage is part of a novel phage lineage among the Siphoviridae family of phages that infect strains of <i>Klebsiella</i>. Based on its features, the vB_KleS-HSE3 phage has potential for controlling infections caused by multidrug-resistant <i>K. pneumoniae.</i>
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