[Background] Klebsiella pneumoniae is a ubiquitous opportunistic bacterium. The high prevalence of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae has propelled the need to explore alternative antibacterial therapies. [Objective] The present study focused on the isolation and characterization of a novel virulent Klebsiella pneumoniae extended-spectrum beta-lactamases-producing phage isolated from sewage therapy. Moreover, we used the mouse model of septicemia to examine the efficacy of phage therapy in treating infections caused by K. pneumoniae produced ESBLs. [Methods] The morphology of the Klebsiella phage F20 was observed with electron microscopy. One-step growth kinetics, host range, and pharmacokinetics of the phage were determined. In additional, F20 was used to rescue mice from bacteremia caused by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae KP-20 in vivo experiments. [Results] F20 exhibited lytic activity evident in clear areas on the bacterial lawns. Morphologically, F20 was classified as a member of the Siphoviridae family and the Caudovirales order. The phage is highly infectious with a short latent period (18 min) and a large burst size (89 PFU/cell). The phage is stable over a wide pH range (5.0 to 9.0) and at high temperatures (50 °C). Administration of F20 after KP-20 challenge significantly decreased the bacterial burden in the blood and organs (lung, liver, spleen and kidney) of mice, and bacterial titers significantly decreased by 1?3 orders of magnitude in mice treated with phage therapy (P<0.001). Administration of F20 can rescue 87.5% of the mice and have no adverse effect, but the control group resulted in a 0 survival rate within 1 day. Phage treatment can significantly improve the survival of mice (P<0.001). [Conclusion] Our study provides the experimental evidence that F20 shows significant treatment efficacy against Klebsiella pneumoniae infection in mice without any adverse effects. The characteristic of the phage F20 greatly increase its utility as biological bactericide.