Polymyxins resistance has emerged worldwide and is threatening the treatment efficacy of multidrug resistant Klebsiella pneumoniae in humans and animals. In this research, we employed whole-genome sequencing (WGS) to investigate the polymyxin B resistance mechanism in selected polymyxin B-susceptible and polymyxin B-resistant K. pneumoniae, isolated from one patient of Huashan Hospital affiliated to Fudan University. The WGS results showed that the two K. pneumoniae all belong to ST11. The average nucleotide identity between the two K. pneumoniae was nearly 100%. No sense mutations of polymyxins resistance associated genes (pmrA, pmrB, phoP, mgrB) were observed in polymyxin B-resistant K. pneumonia (PRKP) compared to the polymyxin B-susceptible isolate. A 42 bp deletion was found in the sequence of phoQ in PRKP. The deletion of amino acid occurred on the periplasmic domain of PhoQ protein. We speculate that this is the domain that MgrB protein interact with the PhoQ protein and negatively regulate the PhoP/PhoQ system. qRT-PCR analysis revealed an overexpression of the pmrA (6.8-fold), pmrB (151.9-fold), pmrC (14.5-fold), pmrK (287.9-fold), phoP (14.5-fold), and phoQ (16.8-fold) genes in the polymyxin B-resistant isolate compared to the expression of the polymyxin B-susceptible K. pneumoniae isolate, suggesting that the phoQ deletion maybe responsible for the increased expression levels of those genes. In conclusion, this study identified a 42 bp deletion in the sequence of phoQ as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to polymyxin B.